Molecules with specificity for cd45 and cd79

ABSTRACT

The present disclosure relates to a multispecific molecule comprising a binding domain specific to the antigen CD45 and a binding domain specific to the antigen CD79a and/or CD79b, compositions comprising same and use of each in treatment, for example treatment of autoimmune disease.

FIELD OF INVENTION

The present disclosure relates to a molecule which is at leastbispecific to the antigens CD45 and CD79, a formulation comprising saidmolecule and use of any one of the same in treatment. The presentdisclosure also extends to methods of preparing said molecules and saidformulations. In an independent aspect the disclosure also extends tonovel antibody sequences and fragments described herein.

BACKGROUND OF INVENTION

Biological mechanisms in vivo are extremely complicated cascades ofsignals, which are difficult to deconvolute and understand. An exampleof such signalling is that required to activate B-cells. The B cellantigen receptor (BCR) is composed of membrane immunoglobulin (mIg)molecules and associated Igα/Igβ (CD79a/CD79b) heterodimers (α/β). ThemIg subunits bind antigen, resulting in receptor aggregation, while theα/β subunits transduce signals to the cell interior. BCR aggregationrapidly activates the Src family kinases Lyn, Blk, and Fyn as well asthe Syk and Btk tyrosine kinases. This initiates the formation of a‘signalosome’ composed of the BCR, the aforementioned tyrosine kinases,adaptor proteins such as CD19 and BLNK, and signaling enzymes such asPLCγ2, PI3K, and Vav.

Signals emanating from the signalosome activate multiple signalingcascades that involve kinases, GTPases, and transcription factors. Thisresults in changes in cell metabolism, gene expression, and cytoskeletalorganization. The complexity of BCR signaling permits many distinctoutcomes, including survival, tolerance (anergy) or apoptosis,proliferation, and differentiation into antibody-producing cells ormemory B cells. The outcome of the response is determined by thematuration state of the cell, the nature of the antigen, the magnitudeand duration of BCR signaling, and signals from other receptors such asCD40, the IL-21 receptor, and BAFF-R.

Many other transmembrane proteins, some of which are receptors, modulatespecific elements of BCR signaling. A few of these, including CD45,CD19, CD22, PIR-B, and FcγRIIB1 (CD32). The magnitude and duration ofBCR signaling are limited by negative feedback loops including thoseinvolving the Lyn/CD22/SHP-1 pathway, the Cbp/Csk pathway, SHIP, Cbl,Dok-1, Dok-3, FcγRIIB1, PIR-B, and internalization of the BCR.

In vivo, B cells are often activated by antigen-presenting cells thatcapture antigens and display them on their cell surface. Activation of Bcells by such membrane-associated antigens requires BCR-inducedcytoskeletal reorganization.

Autoreactive B cells are responsible for the production of pathogenicautoantibodies which can either directly or indirectly cause orexacerbate autoimmune conditions. Depletion of CD20 positive B cells hasbeen used to successfully treat a number of autoimmune conditions andthus established conclusively that B cells play an important role incausing or maintaining a number of autoimmune diseases. Although B celldepletion has been a successful therapeutic option evidence also existsthat control of B cell growth and activation status can also be aneffective way to modulate B cell function. Alternative strategies thatdo not deplete B cells and offer the flexibility of controlling B cellswithout long term suppression of B cell immunity which has been shown tobe associated with some side effects would therefore be desirable. Inaddition not all B cell responses or activities are harmful and evidencesuggests that maintenance of regulatory B cell populations can beprotective. Such an approach should be effective in diseases which haveabnormal B cell function caused by inappropriate or excessive BcRsignalling. Examples include, but are not limited to, inflammation,autoimmunity and cancer. Of particular interest are diseases that eitherhave a direct requirement for BcR signalling or require inhibition orstimulation of humoral immune responses.

Bispecific antibodies are widely expected to play a major role in thenext generation of biotherapeutics (D. Holmes, Nature Rev Drug DiscNovember 2011: 10; 798). They have the potential to deliver superior,long term, broad efficacy in a greater proportion of patients. This canbe achieved by either co-engaging different antigens simultaneouslywithin a common disease pathway, thereby reducing redundancy; or bytargeting antigens from independent pathways to provide an additive orsynergistic effect.

To date strategies to inhibit B cell function without deleting the Bcell have focused on exploiting the natural mechanism of regulation byCD32b (FcgRIIB). These include bispecific antibodies to CD79b/CD32b(Veri et al., Arthritis & Rheumatism 2010 62 1933-1943), CD19/CD32b(Karnell et al., J. Immunol 2014 192 1430-1490) and an antibody to CD19with an Fc with enhanced CD32b binding (Chu et al., Arthritis &Rheumatology 2014 66 1153-1164).

Bispecific antibodies are widely expected to play a major role in thenext generation of biotherapeutics (D. Holmes, Nature Rev Drug DiscNovember 2011:10; 798). They have the potential to deliver superior,long term, broad efficacy in a greater proportion of patients. This canbe achieved by either co-engaging different antigens simultaneouslywithin a common disease pathway, thereby reducing redundancy; or bytargeting antigens from independent pathways to provide an additive orsynergistic effect.

To date strategies to inhibit B cell function without deleting the Bcell have focused on exploiting the natural mechanism of regulation byCD32b (FcgRIIB). These include bispecific antibodies to CD79b/CD32b(Arthritis & Rheumatism 2010 62 1933-1943), CD19/CD32b (J. Immunol 2014192 1430-1490) and an antibody to CD19 with an Fc with enhanced CD32bbinding (Arthritis & Rheumatology 2014 66 1153-1164).

Co-ligation of Fc gamma receptor IIb (CD32b) with the B cell receptoroccurs to naturally regulate signalling, in particular when antigen isbound to antibody in small immune complexes. CD32b then recruits thephosphatases SHP-1 and SHIP-1 which antagonise BcR activation. Althoughthis natural regulatory mechanism can control B cell function,disruption of CD32b function caused by variation in the protein sequenceof CD32b can lead to autoimmune disease and this receptor can be downregulated in autoimmune disease—e.g. as in the case of SLE. Alternativeways of blocking B cell activity are thus desirable as they offeralternative, non-natural, ways of regulating BcR function. Thesealternative mechanisms are likely to be particularly important whennatural mechanisms are dis-functional in the given disease.

Bispecific antibodies facilitate access to novel biology such as:

-   -   1) cross-linking receptors on a cell, if appropriate,    -   2) inducing cell mediated effects,    -   3) localizing a cytokine to a cell to regulate signaling or        locally block cytokine function,    -   4) engaging multiple epitopes simultaneously to generate “new        activity”, increase function or specificity, which may not be        exhibited by a single monoclonal antibody or indeed mixtures of        un-linked antibodies (poly-monoclonals), including mixtures        directed to different antigens.

Under normal physiological conditions upon antigen binding CD45 isexcluded from the BcR complex. The present inventors have surprisinglyfound that using a bispecific antibody to couple the BcR (CD79) to themolecule CD45, BCR signalling can be inhibited. Thus by physicallylinking the BcR with CD45 through use of a bispecific antibody theinventors have found that activation in B cells can be inhibited.

The present inventors have therefore identified a synergistic functionfor molecules which are at least bispecific for CD45 and CD79. Thisfunction seems to be detectable primarily when binding regions with thecombination of specificities are provided in a bispecific(multispecific) format, as opposed to simply being provided as a mixtureof, for example monoclonal antibodies or binding fragments thereof.

The multispecific molecules of the invention are therefore useful incontrolling aberrant B cell functions associated with certain diseasessuch as autoimmunity and cancer.

SUMMARY OF THE DISCLOSURE

Thus provided is a multispecific molecule comprising a binding domainspecific to the antigen CD45 and a binding domain specific to theantigen CD79.

The combination according to the present disclosure in a bispecificformat shows interesting biological activity in functional in vitroassays, for example inhibition of B cell signalling as measured by anyone of the following: inhibition of phosphorylation of Akt 5473,inhibition of phosphorylation of P38 and PLCγ2 Y759 inhibition of IkB,in addition to the inhibition of expression of CD86, CD71 and/or CD40 onB cells. The same level of activity is not apparent for individualcomponents alone or the components provided in admixture. However, theactivity is apparent when a bispecific construct with specificity forCD45 and CD79 is provided.

The inhibition of certain B cell functions observed in these assays isindicative that a multispecific molecule of the invention, comprising abinding domain specific to CD45 and a binding domain specific to CD79,may be used to alter B cell function and, for example to provide atherapeutic alternative to depletion of B cells.

B cell receptor signalling is a critical function of the B cell and arequirement for antigen specific activation of B cells. BcR signallingis critical from early stages of B cell development through to theactivation and development of memory B cell responses. The B cellreceptor is composed of a surface immunoglobulin (Ig) molecule whichassociates with heterodimeric complex of CD79a and CD79b. When surfaceIg recognises antigen it is thought that this results in a clustering ofthe CD79a/b complex which results in downstream activation of theimmediate signalling cascade, which includes Src family kinases as wellas Syk and Btk tyrosine kinases. This signalling complex then canrecruit adaptor proteins such as CD19 and BLNK and results in activationof PLCγ2 and PI3K which in turn can activate further downstream pathwayssuch as those that control B cell growth, survival and differentiation.This signalling complex can be further regulated by other second signalsvia signalling through BAFF-R, IL-21R and CD40 and can also be regulatedby other signalling molecules such as CD19, CD21, CD83, CD22, CD32b andCD45 amongst others. Upon recognition of antigen by the BcR one of thefirst responses activated is the up-regulation of surface receptors suchas the co-stimulatory molecules CD80 and CD86. These molecules bind tocorresponding receptors on T cells which deliver further survival andactivation signals that allow survival and expansion of T cells thatrecognise antigen in the context of MHC class II. This response isfurther amplified by the ability of B cells to present antigen in thecontext of MHC class II back to the T cell, which releases factors suchas IL-2 and IL-21. These cytokines in turn expand B cell number greatly.

Furthermore, inhibition of B cell receptor signalling can lead toinhibition of downstream functions. One such outcome would be theinhibition of co-stimulatory molecules such as CD86 (or reducedexpression of the same) which will lead to the inhibition of T cellfunction, survival and differentiation.

Thus inhibition of B cell receptor signalling can be beneficial incontrolling aberrant B cell functions associated with autoimmunity andcancer. B cell receptor signalling is required for B cell proliferation,differentiation, antigen presentation and cytokine release in autoimmunedisease. Thus inhibiting BcR activity can regulate B cell functions suchas immunoglobulin secretion, T cell activation and control inappropriateB cell activity associated with, for example autoimmune conditions. Inaddition there are some B cell leukaemias and lymphomas that require Bcell receptor signalling for survival and growth which may be controlledby inhibitors of B cell receptor activation.

In one embodiment the binding domain or binding domains of themulti-specific molecules of the present invention each independentlycomprise one or two (such as two) antibody variable domains specific toa relevant antigen (such as CD45 or CD79 or a further antigen if themolecule is at least trispecific).

CD79 as used herein refers to the complex composed of CD79a and CD79b.Accordingly, antibodies which bind CD79 may bind to CD79a and/or CD79b.Binds to CD79a and/or CD79b as employed herein refers to: specific toCD79a, specific to CD79b, specific to both CD79a and b (i.e. recognisesan epitope on CD79a and also recognises an epitope on CD79b—i.e. panspecific) or is specific to the complex of CD79a and CD79b (i.e.recognises an epitope formed from the interaction of CD79a and CD79b inthe complex form).

In one embodiment an antibody or binding fragment thereof employed inthe molecules of the present disclosure is specific to CD79a.

In one embodiment an antibody or binding fragment thereof employed inthe molecules of the present disclosure is specific to CD79b.

In one embodiment an antibody or binding fragment thereof employed inthe molecules of the present disclosure is specific to CD79 complex,i.e. it recognises an epitope present in the complex and is specificthereto, for example an epitope comprising an interaction between CD79aand CD79b.

In one embodiment even where the binding domain is specific to CD79a orCD79b it will be appreciated that the binding domain will still bind toCD79a or CD79b when in the complex form.

Where there are two variable regions in a binding domain or in eachbinding domain then the two variable regions will generally workco-operatively to provide specificity for the relevant antigen, forexample they are a cognate pair or affinity matured to provide adequateaffinity such that the domain is specific to a particular antigen.Typically they are a heavy and light chain variable region pair (VH/VLpair).

In one embodiment the molecule of the present disclosure is bispecific.

In one embodiment the molecule of the present disclosure is trispecific,for example where the third binding domain is specific to serum albumin,for example human serum albumin.

In one embodiment the molecule of the present disclosure is monospecificfor CD79 and CD45 i.e. the molecule only comprises one binding domainwhich binds CD79 and one binding domain which binds CD45.

In one embodiment the multispecific molecule of the present disclosureis a single chain.

In one embodiment the multispecific molecule of the present disclosurecomprises a heavy chain and also a light chain. In one example, asemployed herein a heavy and light chain pairing is not referred to as adimer, particularly where in one embodiment the molecule of the presentdisclosure does not comprise multimers, such as dimers of the antibody,unit/fragment or components.

In one aspect, there is provided a multi-specific antibody moleculecomprising or consisting of:

-   -   a) a polypeptide chain of formula (I):

V_(H)—CH₁—X—(V₁)_(p);

-   -   b) a polypeptide chain of formula (II):

V_(L)—C_(L)—Y—(V₂)_(q);

-   -   wherein:    -   V_(H) represents a heavy chain variable domain;    -   CH₁ represents a domain of a heavy chain constant region, for        example domain 1 thereof;    -   X represents a bond or linker, for example an amino acid linker;    -   Y represents a bond or linker, for example an amino acid linker;    -   V₁ represents a dab, scFv, dsscFv or dsFv;    -   V_(L) represents a variable domain, for example a light chain        variable domain;    -   C_(L) represents a domain from a constant region, for example a        light chain constant region domain, such as Ckappa;    -   V₂ represents a dab, scFv, dsscFv or dsFv;    -   p is 0 or 1;    -   q is 0 or 1; and        when p is 1 q is 0 or 1 and when q is 1 p is 0 or 1 i.e. p and q        do not both represent 0.

In one embodiment the molecule comprises no more than one binding sitefor CD45 and no more than one binding site for CD79.

The above format is particularly useful for screening combinations ofvariable regions, for example in longer term assays and for therapeuticuse.

In one embodiment q is 0 and p is 1.

In one embodiment q is 1 and p is 1.

In one embodiment V₁ is a dab and V₂ is a dab and together they form asingle binding domain of a co-operative pair of variable regions, suchas a cognate V_(H)/V_(L) pair.

In one embodiment V_(H) and V_(L) are specific to, CD79, for exampleCD79a or CD79b.

In one embodiment the V₁ is specific to, CD79, for example CD79a.

In one embodiment the V₂ is specific to, CD79, for example CD79a.

In one embodiment the V₁ is specific to, CD79, for example CD79b.

In one embodiment the V₂ is specific to, CD79, for example CD79b.

In one embodiment the V₁ and V₂ together (eg as one binding domain) arespecific to, CD79, for example CD79a or CD79b.

In one embodiment V_(H) and V_(L) are specific to, CD45.

In one embodiment the V₁ is specific to, CD45.

In one embodiment the V₂ is specific to, CD45.

In one embodiment the V₁ and V₂ together (eg as one binding domain) arespecific to, CD45.

In one embodiment the molecule of the present disclosure is or comprisesa fusion protein.

In one embodiment there is provided a multispecific molecule accordingto the present disclosure, which is a bispecific protein complex havingthe formula A-X:Y-B wherein:

-   -   A-X is a first fusion protein;    -   Y-B is a second fusion protein;    -   X:Y is a heterodimeric-tether;    -   A comprises a first binding domain specific to CD45, CD79a,        CD79b or a complexed of CD79a and b;    -   B comprises a second binding domain specific to CD45, CD79a,        CD79b or a complexed of CD79a and b;    -   X is a first binding partner of a binding pair;    -   Y is a second binding partner of the binding pair; and    -   : is an interaction (such as a binding interaction) between X        and Y, and    -   wherein at least one of A or B is specific to CD45 and the other        is specific to CD79a, CD79b or a complexed form thereof.

The above format is a convenient because it provides a rapid andefficient way of assembling bispecific formats that, for example can besubjected to in vitro testing in functional assays. This may facilitatethe choice of a preferred pair of variable regions, which maysubsequently be incorporated into an alternative, therapeuticmultispecific antibody format.

Whilst not wishing to be bound by theory different permutations ofvariable regions specific to CD45 combined with a range of variableregions specific to CD79 may give access to different nuances inbiological function.

The invention also provides novel CD45 antibodies, for example for usein the multispecific molecules of the present invention or forincorporation into any other suitable antibody format. The inventionalso provides novel CD79 antibodies, for example for use in themultispecific molecules of the present invention or for incorporationinto any other suitable antibody format.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a bar chart of the relative potency of inhibition ofphosphorylated Akt for bispecific and bivalent combinations ofantibodies with specificity for CD45 and CD79b.

FIG. 2 is a bar chart of the relative potency of inhibition ofphosphorylated PLCg2 for bispecific and bivalent combinations ofantibodies with specificity for CD45 and CD79b.

FIG. 3 is a graph showing the titration of the effect of the bispecificcombination of CD45 and CD79b on CD86 expression on anti-IgM stimulatedB cells.

FIG. 4 is a graph of inhibition of phosphorylated PLCg2 for bispecificproteins with specificity for CD45 and CD79b with different V regions

FIG. 5 is an extract from Chan and Carter Reviews Immunology vol 10, May2010, 301 showing certain antibody formats.

FIG. 6 shows data for the antigen grid cross specificities. Antigen2=CD79b and antigen 4=CD45. Values are percentage inhibition (negativevalue for activation) of phosphorlylation of Syk & represent the mean ofmultiple V region combinations evaluated.

FIG. 7 shows data for the antigen grid cross specificities. Antigen2=CD79b and antigen 4=CD45. Values are percentage inhibition (negativevalue for activation) of PLCγ2 & represent the mean of multiple V regioncombinations evaluated.

FIG. 8 shows data for the antigen grid cross specificities. Antigen2=CD79b and antigen 4=CD45Values are percentage inhibition (negativevalue for activation) of AKT & represent the mean of multiple V regioncombinations evaluated.

FIG. 9 shows the percentage inhibition of the phosphorlylation of Syk,PLCγ2 & AKT for each V-region combination for CD79b specificity in Fab-Xcombined with CD45 specificity in Fab-Y.

FIG. 10 shows the percentage inhibition of the phosphorlylation of Syk,PLCγ2 & AKT for each V-region combination for CD79b specificity in Fab-Ycombined with CD45 specificity in Fab-X

FIGS. 11 & 12 shows inhibition of PLCγ2 (+/−SD) by purified CD79b-CD45(transiently expressed) on IgM stimulated B-cells from donor 129 & 130

FIGS. 13 & 14 shows inhibition of p38 (+/−SD) by purified CD79b-CD45(transiently expressed) on IgM stimulated B-cells from donor 129 & 130

FIGS. 15 & 16 shows inhibition of Akt (+/−SD) by purified CD79b-CD45(transiently expressed) on IgM stimulated B-cells from donor 129 & 130

FIG. 17 shows the inhibition of tetanus toxoid IgG production from PBMCscultured with different multispecific molecules

DETAILED DESCRIPTION OF THE DISCLOSURE

“Multispecific molecule” as employed herein refers to a molecule withthe ability to specifically bind at least two distinct antigens, forexample different antigens. In one embodiment the multispecific moleculeis a bispecific, trispecific or tetraspecific molecule, in particular abispecific molecule or trispecific molecule.

Thus in one aspect the disclosure extends to a molecule of a suitableformat specific to at least CD45 and CD79a and to use ofantibodies/fragments or combinations thereof specific to CD45 and CD79ain a multispecific molecule, such as a bispecific format or trispecificformat.

Thus in one aspect the disclosure extends to a molecule of a suitableformat specific to at least CD45 and CD79b and to use ofantibodies/fragments or combinations thereof specific to CD45 and CD79bin a multispecific molecule, such as a bispecific format or trispecificformat.

Thus in one aspect the disclosure extends to a molecule of a suitableformat specific to at least CD45 and CD79a/b complex and to use ofantibodies/fragments or combinations thereof specific to CD45 andCD79a/b complex in a multispecific molecule, such as a bispecific formator trispecific format.

In one embodiment the molecule of the present disclosure is trispecific,for example where the third binding domain is capable of extending thehalf-life of the molecule, for example by binding a serum carrierprotein.

A variety of proteins exist in plasma and include thyroxine-bindingprotein, transthyretin, α1-acid glycoprotein, transferrin, fibrinogenand albumin, or a fragment of any thereof (Bartalena & Robbins, 1993,Clinics in Lab. Med. 13:583-598; Bree et al., 1986, Clin. Pharmacokin.11:336-342; Gitlin et al. 1964, J. Clin. Invest. 10:1938-1951; Peters,1985, Adv Protein Chem. 37:161-245; Waldeman & Strober, 1969, Progr.Allergy, 13:1-110. In on example the third binding domain is specific toserum albumin, for example human serum albumin.

Multispecific Molecule Formats

Examples of suitable multispecific molecules are known in the art, forexample as disclosed in the review “The coming of Age of EngineeredMultivalent Antibodies, Nunez-Prado et al Drug Discovery Today Vol 20Number 5 Mar. 2015, page 588-594, D. Holmes, Nature Rev Drug DiscNovember 2011:10; 798, Chan and Carter, Nature Reviews Immunology vol10, May 2010, 301 incorporated herein by reference.

In one embodiment multispecific formats include those known in the artand those described herein, such as wherein the molecule format isselected from the group comprising or consisting of:

-   -   tandem sdAb, tandem sdAb-sdAb (three sdAbs);    -   (scFv)₂ (also referred to as tandem scFv), scFv-dsFv,        dsscFv-dsFv (dsFv)₂;    -   diabody, dsdiabody, didsdiabody,    -   scdiabody, dsscdiabody, didsscdiabody;    -   Dart antibody i.e, VL₁ linker VH₂ linker and VH₁ linker VL₂        wherein the C-terminus of VH₁ and VH₂ are joined by a disulfide        bond;    -   BITE, dsBiTE, didsBiTE;    -   Di-diabody (see Nunez-Prado et al in particular molecule number        25 in FIG. 1 therein), dsdi-diabody, didsdi-diabody;    -   triabody, dstriabody, didstriabody, tridstriabody;    -   tetrabodies, dstetrabody, didstetrabody, tridstetrabody,        tetradstetrabody;    -   tandab (see Nunez-Prado et al in particular molecule number 22        in FIG. 1 therein); dstandab, didstandab, tridstandab,        tetradstandab;    -   [sc(Fv)₂]₂, (see Nunez-Prado et al in particular molecule number        22 in FIG. 1 therein), ds[sc(Fv)₂]₂, dids[sc(Fv)₂]₂,        trids[sc(Fv)₂]₂, tetrads[sc(Fv)₂]₂,    -   Pentabody (see Nunez-Prado et al in particular molecule number        27 in FIG. 1 therein);    -   Fab-scFv (also referred to as a bibody), Fab′scFv, FabdsscFv (or        BYbe), Fab′dsscFv;    -   tribody, dstribody, didstribody (also referred to as FabdidsscFv        or TrYbe or Fab-(dsscFv)₂), Fab′didsscFv;    -   Fabdab, FabFv, Fab′dab, Fab′Fv;    -   Fab single linker Fv (also referred to herein as FabdsFv as        disclosed in WO2014/096390), Fab′ single linker Fv (also        referred to herein as Fab′dsFv);    -   FabscFv single linker Fv, Fab′scFv single linker Fv;    -   FabdsscFv single linker Fv, Fab′dsscFv single linker Fv;    -   FvFabFv, FvFab′Fv, dsFvFabFv, dsFvFab′Fv, FvFabdsFv, FvFab′dsFv,        dsFvFabdsFv, dsFvFab′dsFv,    -   FabFvFv, Fab′FvFv, FabdsFvFv, Fab′ dsFvFv, FabFvdsFv,        Fab′FvdsFv, FabdsFvdsFv, Fab′ dsFvdsFv,    -   diFab, diFab′ including a chemically conjugated diFab′,    -   (FabscFv)₂, (Fab)₂scFvdsFv, (Fab)₂dsscFvdsFv, (FabdscFv)₂,    -   (Fab′scFv)₂, (Fab′)₂scFvdsFv, (Fab′)₂dsscFvdsFv, (Fab′dscFv)₂,    -   V_(H)HC_(K) (see Nunez-Prado et al in particular molecule number        6 in FIG. 1 therein);    -   minibody, dsminibody, didsminibody,    -   a miniantibody (ZIP) [see Nunez-Prado et al in particular        molecule number 7 in FIG. 1 therein], dsminiantibody (ZIP) and        didsminiantibody (ZIP);    -   tribi-minibody [see Nunez-Prado et al in particular molecule        number 15 in FIG. 1 therein] dstribi-minibody,        didstribi-minibody, tridstribi-minibody;    -   diabody-CH₃, dsdiabody-CH₃, didsdiabody-CH₃, scdiabody-CH₃,        dsscdiabody-CH₃, didsscdiabody-CH₃,    -   tandemscFv-CH₃, tandemdsscFv-CH₃, tandemdidsscFv-CH₃,        tandemtridsscFv-CH₃, tandemtetradsscFv-CH₃,    -   scFv-Fc (also referred to herein as a (scFvCH₂CH₃)₂) as        described in WO2008/012543 and a single chain version thereof,        dsscFvscFv-Fc, dsscFv-Fc (also referred to herein as        (dsscFvCH₂CH₃)₂), scFv-dsFv-Fc, dsscFv-dsFv-Fc, dsFv-Fc (also        referred to herein a (dsFvCH₂CH₃)₂),    -   scorpion molecule (Trubion) i.e. a binding domain, linker        —CH₂CH₃ binding domain as described in U.S. Pat. No. 8,409,577;    -   SMIP (Trubion) i.e. (scFv-CH₂CH₃)₂;    -   (dsFvCH₂CH₃)₂, tandem scFv-Fc, tandem dsscFvscFv-Fc, tandem        dsscFv-Fc,    -   scFv-Fc-scFv, dsscFv-Fc-scFv, scFv-Fc-dsscFv,    -   diabody-Fc, dsdiabody-Fc, didsdiabody-Fc, triabody-Fc,        dstriabody-Fc, didstriabody-Fc, tridstriabody-Fc, tetrabody-Fc,        dstetrabody-Fc, didstetrabody-Fc, tridstetrabody-Fc,        tetradstetrabody-Fc, dstetrabody-Fc, didstetrabody-Fc,        tridstetrabody-Fc, tetradstetrabody-Fc, scdiabody-Fc,        dsscdiabody, didsscdiabody;    -   bi or trifunctional antibody, for example with different heavy        chain variable regions and common light chains for example Merus        bispecific antibody format (BICLONICS) with common light chains        of a fixed sequence and different heavy chains (including        different CDRs) and engineered CH₃ domain to drive the        dimerization o the different heavy chains,    -   Duobody (i.e. wherein one full length chain in the antibody has        different specificity to the other full length chain in the        antibody);    -   a full-length antibody wherein Fab arm exchange has been        employed to create a bispecific format;    -   bi or tri functional antibody, wherein a full-length antibody        has common heavy chain and different light chains also referred        to as kappa/lambda body’ or ‘κ/λ-body, see for example        WO2012/023053 incorporated herein by reference;    -   Ig-scFv one, two, three or four from the C terminus of heavy or        light chain, scFv-Ig one, two, three or four from the N terminus        of heavy or light chain, single linker Ig-Fv, Ig-dsscFv one,        two, three or four from the C terminus of heavy or light chain        (with one, two, three or four disulfide bonds);    -   Ig-dsscFv one, two, three or four from the N terminus of heavy        or light chain (with one, two, three or four disulfide bonds),    -   Ig single linker Fv (see PCT/EP2015/064450),    -   Ig-dab, dab-Ig, scFv-Ig, V-Ig, Ig-V,    -   scFabFvFc, scFabdsFvFc (single linker version scFavFv),        (FabFvFc)₂, (FabdsFvFc)₂, scFab′FvFc, scFab′ dsFvFc,        (Fab′FvFc)₂, (Fab′ dsFvFc)₂ and    -   DVDIg, which are discussed in more detail below.

In one embodiment multispecific molecule formats include those known inthe art and those described herein, such as wherein the molecule formatis selected from the group comprising or consisting of: diabody,scdiabody, triabody, tribody, tetrabodies, tandem scFv, FabFv, Fab′Fv,FabdsFv, Fab-scFv, Fab-dsscFv, Fab-(dsscFv)₂, diFab, diFab′, tandemscFv-Fc, scFv-Fc-scFv, scdiabody-Fc, scdiabody-CH₃, Ig-scFv, scFv-Ig,V-Ig, Ig-V, Duobody and DVDIg, which are discussed in more detail below.

In one embodiment the multispecific antibody molecule of the presentdisclosure does not comprise an Fc domain i.e. does not comprise a CH2and CH3 domain, for example the molecule is selected from the groupcomprising a tandem scFv, scFv-dsFv, dsscFv-dsFv didsFv, diabody,dsdiabody, didsdiabody, scdiabody (also referred to as an (scFv)2),dsscdiabody, triabody, dstriabody, didstriabody, tridstriabody,tetrabodies, dstetrabody, didstetrabody, tridstetrabody,tetradstetrabody, tribody, dstribody, didstribody, Fabdab, FabFv,Fab′dab, Fab′Fv, Fab single linker Fv (as disclosed in WO2014/096390),Fab′ single linker Fv, FabdsFv, Fab′dsFv, Fab-scFv (also referred to asa bibody), Fab′scFv, FabdsscFv, Fab′dsscFv, FabdidsscFv, Fab′didsscFv,FabscFv single linker Fv, Fab′scFv single linker Fv, FabdsscFvs singlelinker Fv, Fab′dsscFv single linker Fv, FvFabFv, FvFab′Fv, dsFvFabFv,dsFvFab′Fv, FvFabdsFv, FvFab′dsFv, dsFvFabdsFv, dsFvFab′dsFv, FabFvFv,Fab′FvFv, FabdsFvFv, Fab′dsFvFv, FabFvdsFv, Fab′FvdsFv, FabdsFvdsFv,Fab′dsFvdsFv, diFab, diFab′ including a chemically conjugated diFab′,(FabscFv)₂, (Fab)₂scFvdsFv, (Fab)₂dsscFvdsFv, (FabdscFv)₂, minibody,dsminibody, didsminibody, diabody-CH₃, dsdiabody-CH₃, didsdiabody-CH₃,scdiabody-CH₃, dsscdiabody-CH₃, didsscdiabody-CH₃, tandemscFv-CH₃,tandemdsscFv-CH₃, tandemdidsscFv-CH₃, tandemtridsscFv-CH₃ andtandemtetradsscFv-CH₃.

In one embodiment the molecule of the present disclosure does notcomprise an Fc domain.

In one embodiment the molecule of the present disclosure comprises analtered Fc domain as described herein below.

Fc domain as employed herein generally refers to —(CH₂CH₃)₂, unless thecontext clearly indicates otherwise.

In one embodiment the molecule of the present disclosure does notcomprise a —CH₂CH₃ fragment.

In one embodiment the molecule of the present disclosure does notcomprise a CH₂ domain.

In one embodiment the molecule of the present disclosure does notcomprise a CH₃ domain.

Molecule as employed herein is used in the biochemistry sense to referto a group of atoms that form an organic, in particular proteinaceousmass, which includes a complex suitable for handling as a single entityunder appropriate conditions once the complex has been formed, forexample a complex formed by two or more polypeptide chains.

Molecule and construct are used interchangeably herein, unless thecontext indicates otherwise. Although, construct may be employed moreoften to refer to a polynucleotide molecule and molecule may be employedmore often to refer an entity primarily comprising an amino acidsequence.

Specificity (or specific) as employed herein refers to where thepartners in the interaction only recognise each other or havesignificantly higher affinity for each other in comparison tonon-partners, for example at least 2, 3, 4, 5, 6, 7, 8, 9, 10 timeshigher affinity, than for example a background level of binding orbinding to another unrelated protein.

A ‘binding domain’ as employed herein refers to a binding region,typically a polypeptide, capable of binding a target antigen, forexample with sufficient affinity to characterise the domain as specificfor the antigen.

Any suitable binding domains may be used in the multispecific moleculesof the present invention. These may be derived from any suitable source.

In one embodiment a biocompatible framework structure is used in abinding domain of the molecules of the present disclosure and suchstructures are based on protein scaffolds or skeletons other thanimmunoglobulin domains. For example, those based on fibronectin,ankyrin, lipocalin, neocarzinostain, cytochrome b, CP1 zinc finger,PST1, coiled coil, LACI-D1, Z domain and tendramisat domains may be used(See for example, Nygren and Uhlen, 1997, Current Opinion in StructuralBiology, 7, 463-469).

The term ‘multi-specific molecules’ as used herein may also includebinding agents based on biological scaffolds including Adnectins,Affibodies, Darpins, Phylomers, Avimers, Aptamers, Anticalins,Tetranectins, Microbodies, Affilins and Kunitz domains.

The multispecific molecule of the present invention is typically amultispecific antibody molecule, ie. at least one or more of the bindingdomains of the multispecific molecule are derived from an antibody orfragment thereof.

Where the binding domain is derived from an antibody, a “binding domainor site” as employed herein is the part of the antibody that contactsthe antigen. In one embodiment the binding domain contains at least onevariable domain or a derivative thereof, for example a pair of variabledomains or derivatives thereof, such as a cognate pair of variabledomains or a derivative thereof. Typically this is a VH/VL pair.

Variable regions (also referred to herein as variable domains) generallycomprise 3 CDRs and a suitable framework. In one embodiment the bindingdomain comprises two variable regions, a light chain variable region anda heavy chain variable region and together these elements contribute tothe specificity of the binding interaction of the antibody or bindingfragment.

A “cognate pair” as employed herein refers to a heavy and light chainpair of variable domains (or a derivative thereof, such as a humanisedversion thereof) isolated from a host as a pre-formed couple. Thisdefinition does not include variable domains isolated from a library,wherein the original pairing from a host is not retained. Cognate pairsmay be advantageous because they are often affinity matured in the hostand therefore may have higher affinity for the antigen to which they arespecific, than a combination of variable domain pairs selected from alibrary, such as phage library.

A “derivative of a naturally occurring domain” as employed herein isintended to refer to where one, two, three, four or five amino acids ina naturally occurring sequence have been replaced or deleted, forexample to optimize the properties of the domain such as by eliminatingundesirable properties but wherein the characterizing feature(s) of thedomain is/are retained. Examples of modifications are those to removeglycosylation sites, GPI anchors, or solvent exposed lysines. Thesemodifications can be achieved by replacing the relevant amino acidresidues with a conservative amino acid substitution.

Other modification in the CDRs may, for example include replacing one ormore cysteines with, for example a serine residue. Asn can be thesubstrate for deamination and this propensity can be reduced byreplacing Asn and/or a neighbouring amino acid with an alternative aminoacid, such as a conservative substitution. The amino acid Asp in theCDRs may be subject to isomerization. The latter can be minimized byreplacing Asp and/or a neighboring amino acid with an alternative aminoacid, for example a conservative substitution.

Humanised versions of a variable region are also a derivative thereof,in the context of the present specification. Humanisation may includethe replacement of a non-human framework for a human framework andoptionally the back-mutation of one or more residues to “donorresidues”. Donor residues as employed herein refers to residues found inthe original variable region isolated from the host, in particularreplacing a given amino acid in the human framework with the amino acidin the corresponding location in the donor framework.

In one embodiment, the binding domain or each binding domain is part of(included or incorporated in) an antibody or an antibody fragment.

In one embodiment the binding domains in the molecules of the presentdisclosure are in immunoglobulin/antibody molecules.

As used herein “antibody molecule” includes antibodies and bindingfragments thereof.

In one embodiment the term “antibody” as used herein refers to animmunoglobulin molecule capable of specific binding to a target antigen,such as a carbohydrate, polynucleotide, lipid, polypeptide, peptideetc., via at least one antigen recognition site (also referred to as abinding site or binding domain herein), located in the variable regionof the immunoglobulin molecule. “Antibody fragments” as employed hereinrefer to antibody binding fragments including but not limited to Fab,modified Fab, Fab′, modified Fab′, F(ab′)2, Fv, single domainantibodies, scFv, Fv, bi, tri or tetra-valent antibodies, Bis-scFv,diabodies, triabodies, tetrabodies and epitope-binding fragments of anyof the above (see for example Holliger and Hudson, 2005, Nature Biotech.23(9):1126-1136; Adair and Lawson, 2005, Drug Design Reviews—Online2(3), 209-217).

A “binding fragment” as employed herein refers to a fragment capable ofbinding a target peptide or antigen with sufficient affinity tocharacterise the fragment as specific for the peptide or antigen

The methods for creating and manufacturing these antibody fragments arewell known in the art (see for example Verma et al., 1998, Journal ofImmunological Methods, 216:165-181). Other antibody fragments for use inthe present disclosure include the Fab and Fab′ fragments described inWO05/003169, WO05/003170 and WO05/003171. Multi-valent antibodies maycomprise multiple specificities e.g. bispecific or may be monospecific(see for example WO92/22853, WO05/113605, WO2009/040562 andWO2010/035012).

The term “Fab fragment” as used herein refers to an antibody fragmentcomprising a light chain fragment comprising a VL (variable light)domain and a constant domain of a light chain (C_(L)), and a V_(H)(variable heavy) domain and a first constant domain (CH₁) of a heavychain.

The Fv refers to two variable domains, for example co-operative variabledomains, such as a cognate pair or affinity matured variable domains,i.e. a V_(H) and V_(L) pair.

Co-operative variable domains as employed herein are variable domainsthat complement each other and/or both contribute to antigen binding torender the Fv (V_(H)/V_(L) pair) specific for the antigen in question.

-   “Single domain antibody” (also referred to herein as a dab and sdAb)    as used herein refers to an antibody fragment consisting of a single    monomeric variable antibody domain. Examples of single domain    antibodies include V_(H) or V_(L) or V_(H)H.-   Tandem-sdAb as employed herein refers to two domain antibodies    connected by a linker, for example a peptide linker, in particular    where the domain antibodies have specificity for different antigens.-   Tandem-sdAb-sdAb as employed herein refers to three domain    antibodies connected in series by two linkers, for example peptide    linkers, in particular where the domain antibodies have specificity    for different antigens.-   dsFv as employed herein refers to an Fv with an intra-variable    disulfide bond. The dsFv may be a component of a larger molecule,    for example the one of the variable domains may be linked, for    example via an amino acid linker to another antibody    fragment/component.-   (dsFv)₂ as employed herein refers to a dsFv with one domain linked,    for example via a peptide linker or a disulfide bond (for example    between, the C-terminus of two V_(H)'s) to a domain in a second    dsFv, the format resembles a (scFv)₂ described below but each pair    of variable regions comprise a intra-variable region disulfide bond.-   Component as employed herein refers to a building block or portion    of a multispecific molecule of the present disclosure, in particular    where the component is an antibody fragment such as scFv, Fab or    other fragment, in particular as described herein.-   Single-chain Fv or abbreviated as “scFv”, as used herein refers to    an antibody fragment that comprises V_(H) and V_(L) antibody domains    linked (for example by a peptide linker) to form a single    polypeptide chain. The constant regions of the heavy and light chain    are omitted in this format.-   dsscFv as employed herein refers to scFv with an intra-variable    region disulfide bond.-   Tandem scFv (also referred to herein as a discFv or (scFv)₂)) as    employed herein refers to two scFvs linked via a single linker such    that there is a single inter-Fv linker, for example as shown in FIG.    5 b.-   Tandem dsscFv (also referred to herein as a scFvdsscFv or    dsscFvscFv) as employed herein refers to two scFvs linked via a    single linker such that there is a single inter-Fv linker, for    example as shown in FIG. 5b , and wherein one of the scFv has an    intravariable region disulfide bond.-   Tandem didsscFv (also referred to herein as a didsscFv) as employed    herein refers to two scFvs linked via a single linker such that    there is a single inter-Fv linker, for example as shown in FIG. 5b ,    and wherein each scFv comprises an intravariable region disulfide    bond.-   scFv-dsFv as employed herein is a scFv linked, for example by a    peptide linker, to an Fv domain which is comprised of two variable    domains linked via a disulfide bond to form a dsFv. In this format    the VH or VL of the scFv may be linked to the VH or VL of the dsFv.-   dsscFv-dsFv as employed herein is a dsscFv linked, for example by a    peptide linker, to an Fv domain which is comprised of two variable    domains linked via a disulfide bond to form a dsFv. In this format    the VH or VL of the dsscFv may be linked to the VH or VL of the    dsFv.-   Diabody as employed herein refers to two Fv pairs V_(H)/V_(L) and a    further V_(H)/V_(L) pair which have two inter-Fv linkers, such that    the V_(H) of a first Fv is linked to the V_(L) of the second Fv and    the V_(L) of the first Fv is linked to the V_(H) of the second Fv.-   dsDiabody as employed herein refers to a diabody comprising an    intra-variable region disulfide bond.-   didsDiabody as employed herein refers to a diabody comprising two    intra-variable region disulfide bonds, i.e. one ds between each pair    of variable regions.-   Sc-diabody as employed herein refers a diabody comprising an    intra-Fv linker, such that the molecule comprises three linkers and    forms two normal scFvs, for example VH₁linkerVL₁ linker VH₂ linker    VL₂-   dssc-diabody as employed herein refers to a sc-diabody with an    intra-variable region disulfide bond.-   didssc-diabody as employed herein refers to a sc-diabody with an    intra-variable region disulfide bond between each pair of variable    regions.-   Dart as employed herein refers to VL₁ linker VH₂ linker and VH₁    linker VL₂ wherein the C-terminous of VH₁ and VH₂ are joined by a    disulfide bond Paul A. Moore et al Blood, 2011; 117(17):4542-4551.-   BITE as employed herein refers to a molecule comprising two pairs of    variable domains in the following format; a domain from pair 1 (eg    VH₁) connected via a linker to a domain from pair 2 (eg VH₂ or VL₂)    said second domain connected by a linker to the further domain from    pair 1 (eg VL₁) in turn connected to the remaining domain from pair    two (i.e VL₂ or VH₂).-   Di-diabody see Nunez-Prado et al in particular molecule number 25 in    FIG. 1 therein.-   Dsdi-diabody as employed herein is a di-diabody with an    intra-variable region disulfide bond.-   Didsdi-diabody as employed herein is a di-diabody with an    intra-variable region disulfide bond between each pair of variable    regions.-   Triabody as employed herein refers to a format similar to the    diabody comprising three Fvs and three inter-Fv linkers.-   dstriabody as employed herein refers to a triabody comprising an    intra-variable region disulfide bond between one of the variable    domain pairs.-   Didstriabody as employed herein refers to a triabody comprising two    intra-variable region disulfide bonds, i.e. one ds between each of    two variable domain pairs.-   Tridstriabody as employed herein refers to a triabody comprising    three intra-variable region disulfide bonds i.e. one ds between each    pair of variable regions.-   Tetrabody as employed herein refers to a format similar to the    diabody comprising four Fvs and four inter-Fv linkers.-   dstetrabody as employed herein refers to a tetrabody comprising an    intra-variable region disulfide bond between one of the variable    domain pairs.-   Didstetrabody as employed herein refers to a tetrabody comprising    two intra-variable region disulfide bonds, i.e. one ds between each    of two variable domain pairs.-   Tridstetrabody as employed herein refers to a tetrabody comprising    three intra-variable region disulfide bonds i.e. one ds between each    of three pairs of variable regions.-   Tetradstetrabody as employed herein refers to a tetrabody comprising    four intra-variable region disulfide bonds i.e. one ds between each    variable domain.-   Tribody (also referred to a Fab(scFv)₂) as employed herein refers to    a Fab fragment with a first scFv appended to the C-terminal of the    light chain and a second scFv appended to the C-terminal of the    heavy the chain.-   dstribody as employed herein refers to a tribody comprising a dsscFv    in one of the two positions.-   didstribody or TrYbe as employed herein refers to a tribody    comprising two dsscFvs.-   dsFab as employed herein refers to a Fab with an intra-variable    region disulfide bond.-   dsFab′ as employed herein refers to a Fab′ with an intra-variable    region disulfide bond.-   scFab is a single chain Fab fragment.-   scFab′ is a single chain Fab′ fragment.-   dsscFab is a dsFab as a single chain.-   dsscFab′ is a dsFab′ as a single chain.-   Fabdab as employed herein refers to a Fab fragment with a domain    antibody appended to the heavy or light chain thereof, optionally    via a linker.-   Fab′ dab as employed herein refers to a Fab′ fragment with a domain    antibody appended to the heavy or light chain thereof, optionally    via a linker.-   FabFv as employed herein refers to a Fab fragment with an additional    variable region appended to the C-terminal of each of the following,    the CH1 of the heavy chain and CL of the light chain see for example    WO2009/040562. The format may be provided as a PEGylated version    thereof see for example WO2011/061492,-   Fab′Fv as employed herein is similar to FabFv, wherein the Fab    portion is replaced by a Fab′. The format may be provided as a    PEGylated version thereof.-   FabdsFv as employed herein refers to a FabFv wherein an intra-Fv    disulfide bond stabilises the appended C-terminal variable regions,    see for example WO2010/035012. The format may be provided as a    PEGylated version thereof.-   Fab single linker Fv and Fab′ single linker as employed herein    refers to a Fab or Fab′ fragment linked to a variable domain, for    example by a peptide linker, and said variable domain is linked to a    second variable domain via an intra-variable domain disulfide bond    thereby forming a dsFv, see for example WO2014/096390.-   Fab-scFv (also referred to as a bibody) as employed herein is a Fab    molecule with a scFv appended on the C-terminal of the light or    heavy chain, optionally via a linker.-   Fab′-scFv as employed herein is a Fab′ molecule with a scFv appended    on the C-terminal of the light or heavy chain, optionally via a    linker.-   FabdsscFv or BYbe as employed herein is a FabscFv with a disulfide    bond between the variable regions of the single chain Fv.-   Fab′dsscFv as employed herein is a Fab′scFv with a disulfide bond    between the variable regions of the single chain Fv.-   FabscFv-dab as employed herein refers to a Fab with a scFv appended    to the C-terminal of one chain and domain antibody appended to the    C-terminal of the other chain.-   Fab′scFv-dab as employed herein refers to a Fab′ with a scFv    appended to the C-terminal of one chain and domain antibody appended    to the C-terminal of the other chain.-   FabdsscFv-dab as employed herein refers to a Fab with a dsscFv    appended to the C-terminal of one chain and domain antibody appended    to the C-terminal of the other chain.-   Fab′dsscFv-dab as employed herein refers to a Fab′ with a dsscFv    appended to the C-terminal of one chain and domain antibody appended    to the C-terminal of the other chain.-   FabscFv single linker Fv as employed herein refers to a Fab single    linker Fv wherein a domain of the Fv is linked to the heavy or light    chain of the Fab and a scFv is linked to the other Fab chain and the    domains of the Fv are connected by an intra-variable region    disulfide.-   FabdsscFv single linker Fv as employed herein refers to a FabscFv    single linker Fv wherein the scFv comprises an intra-variable region    disulfide bond.-   Fab′scFv single linker Fv as employed herein refers to a Fab′ single    linker Fv wherein a domain of the Fv is linked to the heavy or light    chain of the Fab and a scFv is linked to the other Fab chain and the    domains of the Fv are connected by an intra-variable region    disulfide.-   Fab′ dsscFv single linker Fv as employed herein refers to a Fab′scFv    single linker Fv wherein the scFv comprises an intra-variable region    disulfide bond.-   FvFabFv as employed herein refers to a Fab with the domains of a    first Fv appended to the N-terminus of the heavy and light chain of    the Fab and the domains of a second Fv appended to the C-terminus of    the heavy and light chain.-   FvFab′Fv as employed herein refers to a Fab′ with the domains of a    first Fv appended to the N-terminus of the heavy and light chain of    the Fab′ and the domains of a second Fv appended to the C-terminus    of the heavy and light chain.-   dsFvFabFv as employed herein refers to a Fab with the domains of a    first Fv appended to the N-terminus of the heavy and light chain of    the Fab wherein the first Fv comprises an intra-variable region    disulfide bond and the domains of a second Fv appended to the    C-terminus of the heavy and light chain.-   FvFabdsFv as employed herein refers to a Fab with the domains of a    first Fv appended to the N-terminus of the heavy and light chain of    the Fab and the domains of a second Fv appended to the C-terminus of    the heavy and light chain and wherein the second Fv comprises an    intra-variable region disulfide bond.-   dsFvFab′Fv as employed herein refers to a Fab′ with the domains of a    first Fv appended to the N-terminus of the heavy and light chain of    the Fab′ wherein the first Fv comprises an intra-variable region    disulfide bond and the domains of a second Fv appended to the    C-terminus of the heavy and light chain.-   FvFab′dsFv as employed herein refers to a Fab′ with the domains of a    first Fv appended to the N-terminus of the heavy and light chain of    the Fab′ and the domains of a second Fv appended to the C-terminus    of the heavy and light chain and wherein the second Fv comprises an    intra-variable region disulfide bond.-   dsFvFabdsFv as employed herein refers to a Fab with the domains of a    first Fv appended to the N-terminus of the heavy and light chain of    the Fab wherein the first Fv comprises an intra-variable region    disulfide bond and the domains of a second Fv appended to the    C-terminus of the heavy and light chain and wherein the second Fv    also comprises an intra-variable region disulfide bond.-   dsFvFab′dsFv as employed herein refers to a Fab′ with the domains of    a first Fv appended to the N-terminus of the heavy and light chain    of the Fab′ wherein the first Fv comprises an intra-variable region    disulfide bond and the domains of a second Fv appended to the    C-terminus of the heavy and light chain and wherein the second Fv    also comprises an intra-variable region disulfide bond.-   FabFvFv as employed herein refers to a Fab fragment with two pairs    of Fvs appended in series to the C-terminal of the heavy and light    chain, see for example WO2011/086091.-   Fab′FvFv as employed herein refers to a Fab′ fragment with two pairs    of Fvs appended in series to the C-terminal of the heavy and light    chain, see for example WO2011/086091.-   FabdsFvFv as employed herein refers to a Fab fragment with two pairs    of Fvs appended in series to the C-terminal of the heavy and light    chain, see for example WO2011/086091, wherein the first Fv pair    attached directly to the C-terminal comprise an intra-variable    region disulfide bond.-   Fab′dsFvFv as employed herein refers to a Fab′ fragment with two    pairs of Fvs appended in series to the C-terminal of the heavy and    light chain, see for example WO2011/086091, wherein the first Fv    pair attached directly to the C-terminal comprise an intra-variable    region disulfide bond.-   FabFvdsFv as employed herein refers to a Fab fragment with two pairs    of Fvs appended in series to the C-terminal of the heavy and light    chain, wherein the second Fv pair at the “C”-terminal of the    molecule comprise an intra-variable region disulfide bond.-   Fab′FvdsFv as employed herein refers to a Fab′ fragment with two    pairs of Fvs appended in series to the C-terminal of the heavy and    light chain, wherein the second Fv pair at the “C”-terminal of the    molecule comprise an intra-variable region disulfide bond.-   FabdsFvdsFv as employed herein refers to a Fab fragment with two    pairs of Fvs appended in series to the C-terminal of the heavy and    light chain, wherein the first and second Fv pair comprise an    intra-variable region disulfide bond.-   Fab′dsFvdsFv as employed herein refers to a Fab′ fragment with two    pairs of Fvs appended in series to the C-terminal of the heavy and    light chain, wherein the first and second Fv comprise an    intra-variable region disulfide bond.-   DiFab as employed herein refers to two Fab molecules linked via    their C-terminus of the heavy chains.-   DiFab′ as employed herein refers to two Fab′ molecules linked via    one or more disulfide bonds in the hinge region thereof.-   DiFab and DiFab′ molecules include chemically conjugated forms    thereof-   (FabscFv)₂ as employed herein refers to a diFab molecule with two    scFvs appended thereto, for example appended to the C-terminal of    the heavy or light chain, such as the heavy chain.-   (Fab′scFv)₂ as employed herein refers to a diFab′ molecule with two    scFvs appended thereto, for example appended to the C-terminal of    the heavy or light chain, such as the heavy chain.-   (Fab)₂scFvdsFv as employed herein refers to a diFab with a scFv and    dsFv appended, for example one from each of the heavy chain    C-terminal.-   (Fab′)₂scFvdsFv as employed herein refers to a diFab′ with a scFv    and dsFv appended, for example one from each of the heavy chain    C-terminal.-   (Fab)₂dsscFvdsFv, as employed herein refers to a diFab with a dsscFv    and dsFv appended, for example from the heavy chain C-terminal.-   (Fab′)₂dsscFvdsFv as employed herein refers to the a diFab′ with a    dsscFv and dsFv appended, for example from the heavy chain    C-terminal.-   Minibody as employed herein refers to (VL/VH—CH₃)₂.-   dsminibody as employed herein refers to (VL/VH—CH₃)₂ wherein one    VL/VH comprises an intra-variable region disulfide bond.-   didsminibody as employed herein refers to a (dsFv-CH3)₂-   scFv-Fc as employed herein refers to a scFv appended to the    N-terminus of a CH2 domain, for example via a hinge, of constant    region fragment —(CH2CH3), such that the molecule has 2 binding    domains.-   dsscFv-Fc as employed herein refers to a dsscFv appended to the    N-terminus of a CH2 domain and a scFv appended to the N-terminus of    a second CH2 domain, for example via a hinge, of constant region    fragment —(CH2CH3)2, such that the molecule has 2 binding domains.-   didsscFv-Fc as employed herein refers to a scFv appended to the    N-terminus of a CH2 domain, for example via a hinge, of constant    region fragment —(CH2CH3)2, such that the molecule has 2 binding    domains-   Tandem scFv-Fc as employed herein refers to two tandem scFvs,    wherein each one is appended in series to the N-terminus of a CH₂    domain, for example via a hinge, of constant region fragment    —(CH₂CH₃), such that the molecule has 4 binding domains.-   Scdiabody-Fc as employed herein is two scdiabodies, wherein each one    is appended to the N-terminus of a CH2 domain, for example via a    hinge, of constant region fragment —CH₂CH₃.-   ScFv-Fc-scFv as employed herein refers to four scFvs, wherein one of    each is appended to the N-terminus and the C-terminus of both the    heavy and light chain of a —CH2CH3 fragment.-   Scdiabody-CH₃ as employed herein refers to two scdiabody molecules    each linked, for example via a hinge to a CH₃ domain.-   kappa/lambda body’ or ‘κ/λ-body is in the format of a normal IgG    with two heavy chains and two light chains, wherein the two light    chains are different to each other, one is a lambda light chain    (VL-CL) and the other is a kappa light chain (VK-CK). The heavy    chain is identical, even at the CDRs as described in WO2012/023053.-   IgG-scFv as employed herein is a full length antibody with a scFv on    the C-terminal of each of the heavy chains or each of the light    chains.-   scFv-IgG as employed herein is a full length antibody with a scFv on    the N-terminal of each of the heavy chains or each of the light    chains.-   V-IgG as employed herein is a full length antibody with a variable    domain on the N-terminal of each of the heavy chains or each of the    light chains.-   IgG-V as employed herein is a full length antibody with a variable    domain on the C-terminal of each of the heavy chains or each of the    light chains-   DVD-Ig (also known as dual V domain IgG) is a full length antibody    with 4 additional variable domains, one on the N-terminus of each    heavy and each light chain.-   Duobody or ‘Fab-arm exchange’ as employed herein is a bispecific IgG    format antibody where matched and complementary engineered amino    acid changes in the constant domains (typically CH3) of two    different monoclonal antibodies lead, upon mixing, to the formation    of heterodimers. A heavy:light chain pair from the first antibody    will, as a result of the residue engineering, prefer to associate    with a heavy:light chain pair of a second antibody. See for example    WO2008/119353, WO2011/131746 and WO2013/060867

Where one or more pairs of variable regions in the multispecificmolecule of the present invention comprise a disulphide bond between VHand VL this may be in any suitable position such as between two of theresidues listed below (unless the context indicates otherwise Kabatnumbering is employed in the list below). Wherever reference is made toKabat numbering the relevant reference is Kabat et al., 1987, inSequences of Proteins of Immunological Interest, US Department of Healthand Human Services, NIH, USA.

In one embodiment the disulfide bond is in a position selected from thegroup comprising:

-   -   V_(H)37+V_(L)95C see for example Protein Science 6, 781-788 Zhu        et al (1997);    -   V_(H)44+V_(L)100 see for example; Biochemistry 33 5451-5459        Reiter et al (1994); or Journal of Biological Chemistry Vol. 269        No. 28 pp. 18327-18331 Reiter et al (1994); or Protein        Engineering, vol. 10 no. 12 pp. 1453-1459 Rajagopal et al        (1997);    -   V_(H)44+V_(L)105 see for example J Biochem. 118, 825-831 Luo et        al (1995);    -   V_(H)45+V_(L)87 see for example Protein Science 6, 781-788 Zhu        et al (1997);    -   V_(H)55+V_(L)101 see for example FEBS Letters 377 135-139 Young        et al (1995);    -   V_(H)100+V_(L)50 see for example Biochemistry 29 1362-1367        Glockshuber et al (1990);    -   V_(H)100b+V_(L)49;    -   V_(H)98+V_(L) 46 see for example Protein Science 6, 781-788 Zhu        et al (1997);    -   V_(H)101+V_(L)46;    -   V_(H)105+V_(L)43 see for example; Proc. Natl. Acad. Sci. USA        Vol. 90 pp. 7538-7542 Brinkmann et al (1993); or Proteins 19,        35-47 Jung et al (1994),    -   V_(H)106+V_(L)57 see for example FEBS Letters 377 135-139 Young        et al (1995)    -   and a position corresponding thereto in variable region pair        located in the molecule.    -   In one embodiment, the disulphide bond is formed between        positions V_(H)44 and V_(L)100.

“Monospecific” as employed herein refers to the ability to bind a targetantigen only once. Thus is one embodiment the multispecific molecules ofthe present invention are monspecific for each antigen

Thus in one embodiment the binding domains of the multispecificmolecules according to the present disclosure are monospecific. This isadvantageous in some therapeutic applications because the molecules ofthe disclosure are not able to cross-link antigen via binding the targetantigen more than once. Thus in one embodiment bispecific ormultispecific molecules of the present disclosure are not able tocross-link by binding the same target twice in two different locations,for example on the same cell or on two different cells.

Cross-linking, in particular in relation to CD79b on the same cell ordifferent cells can generate signals in vivo, for example whichstimulate the activity of the target antigen

In another embodiment, for example where the molecules of the disclosurecomprise at least three binding domains then two or three bindingdomains (for example antibodies, fragments or a combination of anantibody and a fragment) may have different antigen specificities, forexample binding to two or three different target antigens.

In one example the multispecific molecules of the present inventioncontain no more than one binding domain for CD22 and no more than onebinding domain for CD79. Each binding domain is monospecific.

In one embodiment, each antibody or antibody fragment employed in themulti-specific molecules of the present disclosure is monovalent.

Thus in one embodiment the binding domains of the multispecificmolecules of the present disclosure are monovalent.

Thus in one embodiment the binding domains of the multispecificmolecules of the present disclosure are monovalent and monospecific.

In one embodiment the multispecific molecule of the present disclosureis comprised of two or more monospecific, monovalent binding domainssuch as Fab, Fab′, scFv, VH, VL, VHH, Fv, dsFv, combined or linked inany suitable way to construct a multispecific molecule, for example asdescribed herein above.

Constant Regions

The antibody constant region domains of a multispecific molecule of thepresent disclosure, if present, may be selected having regard to theproposed function of the multispecific antibody molecule, and inparticular the effector functions which may be required. For example,the constant region domains may be human IgA, IgD, IgE, IgG or IgMdomains. In particular, human IgG constant region domains may be used,especially of the IgG1 and IgG3 isotypes when the antibody molecule isintended for therapeutic uses and antibody effector functions arerequired. Alternatively, IgG2 and IgG4 isotypes may be used when theantibody molecule is intended for therapeutic purposes and antibodyeffector functions are not required. It will be appreciated thatsequence variants of these constant region domains may also be used. Forexample IgG4 molecules in which the serine at position 241 has beenchanged to proline as described in Angal et al., 1993, MolecularImmunology, 1993, 30:105-108 may be used. Accordingly, in the embodimentwhere the antibody is an IgG4 antibody, the antibody may include themutation S241P.

In one embodiment, the antibody heavy chain comprises a CH₁ domain andthe antibody light chain comprises a CL domain, either kappa or lambda.

In one embodiment, the antibody heavy chain comprises a CH₁ domain, aCH₂ domain and a CH₃ domain and the antibody light chain comprises a CLdomain, either kappa or lambda.

The four human IgG isotypes bind the activating Fcγ receptors (FcγRI,FcγRIIa, FcγRIIIa), the inhibitory FcγRIIb receptor, and the firstcomponent of complement (C1q) with different affinities, yielding verydifferent effector functions (Bruhns P. et al., 2009. Specificity andaffinity of human Fcgamma receptors and their polymorphic variants forhuman IgG subclasses. Blood. 113(16):3716-25), see also Jeffrey B.Stavenhagen, et al. Cancer Research 2007 Sep. 15; 67(18):8882-90.

Binding of IgG to the FcγRs or C1q depends on residues located in thehinge region and the CH₂ domain. Two regions of the CH₂ domain arecritical for FcγRs and C1q binding, and have unique sequences in IgG2and IgG4. Substitutions into human IgG1 of IgG2 residues at positions233-236 and IgG4 residues at positions 327, 330 and 331 have been shownto greatly reduce ADCC and CDC (Armour K L. et al., 1999. Recombinanthuman IgG molecules lacking Fcgamma receptor I binding and monocytetriggering activities. Eur J Immunol. 29(8):2613-24 and Shields R L. etal., 2001. High resolution mapping of the binding site on human IgG1 forFc gamma RI, Fc gamma RII, Fc gamma RIII, and FcRn and design of IgG1variants with improved binding to the Fc gamma R. J Biol Chem.276(9):6591-604). Furthermore, Idusogie et al. demonstrated that alaninesubstitution at different positions, including K322, significantlyreduced complement activation (Idusogie E E. et al., 2000. Mapping ofthe C1q binding site on rituxan, a chimeric antibody with a human IgG1Fc. J Immunol. 164(8):4178-84). Similarly, mutations in the CH₂ domainof murine IgG2A were shown to reduce the binding to FcγRI, and C1q(Steurer W. et al., 1995. Ex vivo coating of islet cell allografts withmurine CTLA4/Fc promotes graft tolerance. J Immunol. 155(3):1165-74).

In one embodiment the Fc region employed is mutated, in particular amutation described herein. In one embodiment the mutation is to removebinding and/or effector function.

In one embodiment the Fc mutation is selected from the group comprisinga mutation to remove binding of the Fc region, a mutation to increase orremove an effector function, a mutation to increase half-life and acombination of the same.

Some antibodies that selectively bind FcRn at pH 6.0, but not pH 7.4,exhibit a higher half-life in a variety of animal models. Severalmutations located at the interface between the CH₂ and CH₃ domains, suchas T250Q/M428L (Hinton P R. et al., 2004. Engineered human IgGantibodies with longer serum half-lives in primates. J Biol Chem.279(8):6213-6) and M252Y/S254T/T256E+H433K/N434F (Vaccaro C. et al.,2005. Engineering the Fc region of immunoglobulin G to modulate in vivoantibody levels. Nat Biotechnol. 23(10):1283-8), have been shown toincrease the binding affinity to FcRn and the half-life of IgG1 in vivo.

However, there is not always a direct relationship between increasedFcRn binding and improved half-life (Datta-Mannan A. et al., 2007.Humanized IgG1 Variants with Differential Binding Properties to theNeonatal Fc Receptor: Relationship to Pharmacokinetics in Mice andPrimates. Drug Metab. Dispos. 35: 86-94).

IgG4 subclass show reduced Fc receptor (FcγRIIIa) binding, antibodies ofother IgG subclasses generally show strong binding. Reduced receptorbinding in these other IgG subtypes can be effected by altering, forexample replacing one or more amino acids selected from the groupcomprising Pro238, Aps265, Asp270, Asn270 (loss of Fc carbohydrate),Pro329, Leu234, Leu235, Gly236, Gly237, Ile253, Ser254, Lys288, Thr307,Gln311, Asn434 and His435.

In one embodiment a molecule according to the present disclosure has anFc of IgG subclass, for example IgG1, IgG2 or IgG3 wherein the Fc ismutated in one, two or all following positions S228, L234 and/or D265.

In one embodiment the mutations in the Fc region are independentlyselected from S228P, L234A, L235A, L235A, L235E and combinationsthereof.

It may be desired to either reduce or increase the effector function ofan Fc region. Antibodies that target cell-surface molecules, especiallythose on immune cells, abrogating effector functions is required. Insome embodiments, for example for the treatment of autoimmunity,enhanced Fc binding on immune cells by increasing negative Fc receptorbinding (FcgRIIb or CD32b) may be desirable see Stavenhagen J B, et alAdvances in Enzyme Regulation 2007 Dec. 3 and Veri M C, et al. ArthritisRheum, 2010 Mar. 30; 62(7):1933-43. Conversely, for antibodies intendedfor oncology use, increasing effector functions may improve thetherapeutic activity.

Numerous mutations have been made in the CH₂ domain of human IgG1 andtheir effect on ADCC and CDC tested in vitro (Idusogie E E. et al.,2001. Engineered antibodies with increased activity to recruitcomplement. J Immunol. 166(4):2571-5). Notably, alanine substitution atposition 333 was reported to increase both ADCC and CDC. Lazar et al.described a triple mutant (S239D/I332E/A330L) with a higher affinity forFcγRIIIa and a lower affinity for FcγRIIb resulting in enhanced ADCC(Lazar G A. et al., 2006. Engineered antibody Fc variants with enhancedeffector function. PNAS 103(11): 4005-4010). The same mutations wereused to generate an antibody with increased ADCC (Ryan M C. et al.,2007. Antibody targeting of B-cell maturation antigen on malignantplasma cells. Mol. Cancer Ther., 6: 3009 3018). Richards et al. studieda slightly different triple mutant (S239D/I332E/G236A) with improvedFcγRIIIa affinity and FcγRlla/FcγRIIb ratio that mediates enhancedphagocytosis of target cells by macrophages (Richards J O et al 2008.Optimization of antibody binding to Fcgamma RIIa enhances macrophagephagocytosis of tumor cells. Mol Cancer Ther. 7(8):2517-27).

Due to their lack of effector functions, IgG4 antibodies represent asuitable IgG subclass for receptor blocking without cell depletion. IgG4molecules can exchange half-molecules in a dynamic process termedFab-arm exchange. This phenomenon can occur between therapeuticantibodies and endogenous IgG4. The S228P mutation has been shown toprevent this recombination process allowing the design of lessunpredictable therapeutic IgG4 antibodies (Labrijn A F. et al., 2009.Therapeutic IgG4 antibodies engage in Fab-arm exchange with endogenoushuman IgG4 in vivo. Nat Biotechnol. 27(8):767-71). This technology maybe employed to create bispecific antibody molecules.

It will also be understood by one skilled in the art that antibodies mayundergo a variety of post-translational modifications. The type andextent of these modifications often depends on the host cell line usedto express the antibody as well as the culture conditions. Suchmodifications may include variations in glycosylation, methionineoxidation, diketopiperazine formation, aspartate isomerization andasparagine deamidation. A frequent modification is the loss of acarboxy-terminal basic residue (such as lysine or arginine) due to theaction of carboxypeptidases (as described in Harris, R J. Journal ofChromatography 705:129-134, 1995). Accordingly, the C-terminal lysine ofthe antibody heavy chain may be absent.

Affinity

The multispecific molecules of the present invention comprise a bindingdomain specific to the antigen CD45 and a binding domain specific to theantigen CD79a and/or CD79b.

In one embodiment a binding domain employed in the molecules of thepresent disclosure is specific to CD45.

In one embodiment a binding domain employed in the molecules of thepresent disclosure is specific to CD79a.

In one embodiment a binding domain employed in the molecules of thepresent disclosure is specific to CD79b.

In one embodiment a binding domain employed in the molecules of thepresent disclosure is specific to CD79 complex, i.e. it recognises anepitope present in the complex and specific thereto, for example anepitope comprising an interaction between CD79a and CD79b.

CD45 (also known as PTPRC) is a known protein. CD45 is a member of theprotein tyrosine phosphatase (PTP) family. PTPs are known to besignaling molecules that regulate a variety of cellular processesincluding cell growth, differentiation, mitotic cycle, and oncogenictransformation. This PTP contains an extracellular domain, a singletransmembrane segment and two tandem intracytoplasmic catalytic domains,and thus belongs to receptor type PTP. Various isoforms of CD45 exist:CD45RA, CD45RB, CD45RC, CD45RAB, CD45RAC, CD45RBC, CD45RO, CD45R (ABC).CD45RA is located on naive T cells and CD45RO is located on memory Tcells. CD45 splice variant isoforms A, B and C are expresseddifferentially on human B cells. CD45 is a member of the ProteinTyrosine Phosphatase (PTP) family: Its intracellular (COOH-terminal)region contains two PTP catalytic domains, and the extracellular regionis highly variable due to alternative splicing of exons 4, 5, and 6(designated A, B, and C, respectively), plus differing levels ofglycosylation. The CD45 isoforms detected are cell type-, maturation,and activation state-specific. In general the long form of the protein(A, B or C) is expressed on naïve or unactivated B cells and the matureor truncated form of CD45 (RO) is expressed on activated ormature/memory B cells.

The human sequence is available in UniProt entry number P08575 andprovided herein in SEQ ID NO:143, or amino acids 24-1304 of SEQ IDNO:143, lacking the signal peptide. The murine version in UniProt entryP06800. The present disclosure relates to all forms of CD45, from anyspecies. In one embodiment CD45 refers to the human form of the proteinand natural variants and isoforms thereof.

In one embodiment the affinity of the binding domain for CD45 in amolecule of the present disclosure is about 100 nM or stronger such asabout 50 nM, 20 nM, 10 nM, 1 nM, 500 pM, 250 pM, 200 pM, 100 pM orstronger, in particular a binding affinity of 50 pM or stronger. Thebinding domain for CD79 may bind to CD79a and/or CD79b.

CD79a (also known as immunoglobulin alpha and B-cell antigen receptorcomplex-associated protein alpha chain) is a known protein. Expressionof CD79a is restricted to B lymphocytes. The human sequence is availablein UniProt under entry P11912 (SEQ ID NO: and without signal sequenceamino acids 33-226 of SEQ ID NO: 141). The murine version is availablein UniProt under entry 11911. The present disclosure relates to allforms of CD79a from any species, in particular human and any naturalvariants thereof. In one embodiment CD79a refers to the human form ofthe protein.

CD79b (also known as immunoglobulin associated beta and clusterdifferentiation 79B) is a known protein. Expression of CD79b isrestricted to B lymphocytes. The human sequence is available in UniProtunder entry P40259 (SEQ ID NO:142 and without signal sequence aminoacids 29-229 of SEQ ID NO:142). The murine version in UniProt underentry P15530. The present disclosure relates to all forms of CD79b, fromany species, in particular human and any natural variants thereof. Inone embodiment CD79b refers to the human form of the protein.

In one embodiment the binding domain specific to CD79 binds CD79a.

In one embodiment the binding domain specific to CD79 binds CD79b.

In one embodiment the binding domain specific to CD79 binds a complex ofCD79a and CD79b

In one embodiment the affinity of the binding domain for CD79 in amolecule of the present disclosure is about 100 nM or stronger such asabout 50 nM, 20 nM, 10 nM, 1 nM, 500 pM, 250 pM, 200 pM, 100 pM orstronger, in particular a binding affinity of 50 pM or stronger. In oneembodiment the affinity of the binding domain for CD79a in a molecule ofthe present disclosure is about 100 nM or stronger such as about 50 nM,20 nM, 10 nM, 1 nM, 500 pM, 250 pM, 200 pM, 100 pM or stronger, inparticular a binding affinity of 50 pM or stronger.

In one embodiment the affinity of the binding domain for CD79b in amolecule of the present disclosure is about 100 nM or stronger such asabout 50 nM, 20 nM, 10 nM, 1 nM, 500 pM, 250 pM, 200 pM, 100 pM orstronger, in particular a binding affinity of 50 pM or stronger.

It will be appreciated that the affinity of the binding domain for CD45may be the same or different from the affinity of the binding domain forCD79.

In one embodiment, the multi-specific antibody molecules of the presentdisclosure or antibody/fragment components thereof are processed toprovide improved affinity for a target antigen or antigens. Suchvariants can be obtained by a number of affinity maturation protocolsincluding mutating the CDRs (Yang et al., J. Mol. Biol., 254, 392-403,1995), chain shuffling (Marks et al., Bio/Technology, 10, 779-783,1992), use of mutator strains of E. coli (Low et al J. Mol. Biol., 250,359-368, 1996), DNA shuffling (Patten et al Curr. Opin. Biotechnol., 8,724-733, 1997), phage display (Thompson et al., J. Mol. Biol., 256,77-88, 1996) and sexual PCR (Crameri et al Nature, 391, 288-291, 1998).Vaughan et al (supra) discusses these methods of affinity maturation.

Antibodies & Generation of the Same

Binding domains for use in the present invention may be generated by anysuitable method known in the art, for example CDRs may be taken fromnon-human antibodies including commercially available antibodies andgrafted into human frameworks or alternatively chimeric antibodies canbe prepared with non-human variable regions and human constant regionsetc.

Typically the binding domains for use in the present invention arebinding domains derived from antibodies which bind the selected antigen,such as antibodies which bind CD45, CD79a and/or CD79b.

Examples of CD45 and CD79 antibodies are known in the art and these maybe employed directly in the molecules of the present invention orscreened for suitability using the methods described herein, andsubsequently modified if necessary, for example humanised, using themethods described herein. Therapeutic anti-CD45 and anti-CD79 antibodieshave been described in the art, for example anti-CD45 antibodiesdisclosed in US2011/0076270, anti-CD79b antibodies disclosed inWO2014/011521 and WO2015/021089.

Examples of CD45 antibodies include rat monoclonal YTH54, YTH25.4, mousemonoclonal from Miltenyi clone 5B1 and clone 30F11, rat monoclonalYAML568, from BD Bioscience mouse monoclonal clone 2D1 catalog No.347460, from Novus mouse monoclonal antibody 5D3A3 catalog No.NBP2-37293, mouse monoclonal HI30 catalog No. NBP1-79127, mousemonoclonal 4A8A4C7A2 catalog No. NBP1-47428, mouse monoclonal 2B11catalog No. NBP2-32934, rat monoclonal YTH24.5 catalog No. NB100-63828,rabbit monoclonal Y321 catalog No. NB110-55701, mouse monoclonalPD7/26/16 catalog No. NB120-875, from Santa Cruz mouse monoclonal fromclone B8 catalog No. sc-28369, mouse monoclonal from clone F10-89-4catalog No. sc-52490, rabbit monoclonal from clone H-230 catalog No.sc-25590, goat monoclonal from clone N-19 catalog No. sc-1123, mousemonoclonal from clone OX1 catalog No. sc-53045, rat monoclonal (T29/33)catalog No sc-18901, rat monoclonal (YAML 501.4) catalog No. sc65344,rat monoclonal (YTH80.103) catalog No sc-59071, mouse monoclonal (35105)catalog No. sc-53201, mouse monoclonal (35-Z6) catalog No. sc-1178,mouse monoclonal (158-4D3) catalog No. sc-52386, mouse monoclonal toCD45RO (UCH-L1) catalog No. sc-1183, mouse monoclonal to CD45RO (2Q1392)catalog No. sc-70712. CD45 antibodies are also disclosed inWO2005/026210, WO02/072832 and WO2003/048327 incorporated herein byreference.

Commercially available anti-CD79a antibodies include mouse monoclonalLS-B4504 (LSBio) from clone HM57, mouse monoclonal LS-B8330, mousemonoclonal LS-C44954, rabbit monoclonal LS-B9093, mouse monoclonalLS-B8513 from clone JCB117, rabbit monoclonal LS-C210607 from cloneSP18, mouse monoclonal LS-C175441 from clone 5E2, mouse monoclonalLS-C338670 from clone 3D3, mouse monoclonal LS-C88120 from cloneHM47/A9, mouse monoclonal LS-C191714, mouse monoclonal LS-C87592, mousemonoclonal LS-C44955, mouse monoclonal LS-C95934, mouse monoclonalLS-C121584, mouse monoclonal LS-C121585, mouse monoclonal LS-C204347,mouse monoclonal LS-C88122, Abcam mouse monoclonal ab3121 [HM47/A9],rabbit monoclonal ab79414, and rabbit monoclonal ab133483.

Commercially available CD79b antibodies include mouse monoclonal Abcamantibody ab33295, rat monoclonal ab23826, mouse monoclonal ab103422,rabbit monoclonal ab134103, rabbit monoclonal ab134147, and rabbitmonoclonal ab183343.

Such commercially available antibodies may be useful tools in thediscovery of therapeutic antibodies.

The skilled person may generate antibodies for use in the multi-specificmolecules of the invention using any suitable method known in the art.

Antigen polypeptides, for use in generating antibodies for example foruse to immunize a host or for use in panning, such as in phage display,may be prepared by processes well known in the art from geneticallyengineered host cells comprising expression systems or they may berecovered from natural biological sources. In the present application,the term “polypeptides” includes peptides, polypeptides and proteins.These are used interchangeably unless otherwise specified. The antigenpolypeptide may in some instances be part of a larger protein such as afusion protein for example fused to an affinity tag or similar. In oneembodiment the host may be immunised with a cell transfected with therelevant protein or polypeptide, for example co-transfected with CD79aand CD79b.

Antibodies generated against an antigen polypeptide may be obtained,where immunisation of an animal is necessary, by administering thepolypeptides to an animal, preferably a non-human animal, usingwell-known and routine protocols, see for example Handbook ofExperimental Immunology, D. M. Weir (ed.), Vol 4, Blackwell ScientificPublishers, Oxford, England, 1986). Many warm-blooded animals, such asrabbits, mice, rats, sheep, cows, camels or pigs may be immunized.However, mice, rabbits, pigs and rats are generally most suitable.Monoclonal antibodies may be prepared by any method known in the artsuch as the hybridoma technique (Kohler & Milstein, 1975, Nature,256:495-497), the trioma technique, the human B-cell hybridoma technique(Kozbor et al 1983, Immunology Today, 4:72) and the EBV-hybridomatechnique (Cole et al Monoclonal Antibodies and Cancer Therapy, pp77-96, Alan R Liss, Inc., 1985).

Antibodies may also be generated using single lymphocyte antibodymethods by cloning and expressing immunoglobulin variable region cDNAsgenerated from single lymphocytes selected for the production ofspecific antibodies by, for example, the methods described by Babcook,J. et al 1996, Proc. Natl. Acad. Sci. USA 93(15):7843-78481; WO92/02551;WO2004/051268 and WO2004/106377.

The antibodies for use in the present disclosure can also be generatedusing various phage display methods known in the art and include thosedisclosed by Brinkman et al. (in J. Immunol. Methods, 1995, 182: 41-50),Ames et al. (J. Immunol. Methods, 1995, 184:177-186), Kettleborough etal. (Eur. J. Immunol. 1994, 24:952-958), Persic et al. (Gene, 1997 1879-18), Burton et al. (Advances in Immunology, 1994, 57:191-280) andWO90/02809; WO91/10737; WO92/01047; WO92/18619; WO93/11236; WO95/15982;WO95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484;5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908;5,516,637; 5,780,225; 5,658,727; 5,733,743; 5,969,108, andWO20011/30305.

In one example the multi-specific molecules of the present disclosureare fully human, in particular one or more of the variable domains arefully human.

Fully human molecules are those in which the variable regions and theconstant regions (where present) of both the heavy and the light chainsare all of human origin, or substantially identical to sequences ofhuman origin, not necessarily from the same antibody. Examples of fullyhuman antibodies may include antibodies produced, for example by thephage display methods described above and antibodies produced by mice inwhich the murine immunoglobulin variable and optionally the constantregion genes have been replaced by their human counterparts e.g. asdescribed in general terms in EP0546073, U.S. Pat. Nos. 5,545,806,5,569,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429, EP 0438474 andEP0463151.

In one example the binding domains of the multi-specific moleculesaccording to the disclosure are humanised.

Humanised (which include CDR-grafted antibodies) as employed hereinrefers to molecules having one or more complementarity determiningregions (CDRs) from a non-human species and a framework region from ahuman immunoglobulin molecule (see, e.g. U.S. Pat. No. 5,585,089;WO91/09967). It will be appreciated that it may only be necessary totransfer the specificity determining residues of the CDRs rather thanthe entire CDR (see for example, Kashmiri et al., 2005, Methods, 36,25-34). Humanised antibodies may optionally further comprise one or moreframework residues derived from the non-human species from which theCDRs were derived.

As used herein, the term “humanised antibody molecule” refers to anantibody molecule wherein the heavy and/or light chain contains one ormore CDRs (including, if desired, one or more modified CDRs) from adonor antibody (e.g. a murine monoclonal antibody) grafted into a heavyand/or light chain variable region framework of an acceptor antibody(e.g. a human antibody). For a review, see Vaughan et al, NatureBiotechnology, 16, 535-539, 1998. In one embodiment rather than theentire CDR being transferred, only one or more of the specificitydetermining residues from any one of the CDRs described herein above aretransferred to the human antibody framework (see for example, Kashmiriet al., 2005, Methods, 36, 25-34). In one embodiment only thespecificity determining residues from one or more of the CDRs describedherein above are transferred to the human antibody framework. In anotherembodiment only the specificity determining residues from each of theCDRs described herein above are transferred to the human antibodyframework.

When the CDRs or specificity determining residues are grafted, anyappropriate acceptor variable region framework sequence may be usedhaving regard to the class/type of the donor antibody from which theCDRs are derived, including mouse, primate and human framework regions.Suitably, the humanised antibody according to the present invention hasa variable domain comprising human acceptor framework regions as well asone or more of the CDRs provided herein.

Examples of human frameworks which can be used in the present disclosureare KOL, NEWM, REI, EU, TUR, TEI, LAY and POM (Kabat et al supra). Forexample, KOL and NEWM can be used for the heavy chain, REI can be usedfor the light chain and EU, LAY and POM can be used for both the heavychain and the light chain. Alternatively, human germline sequences maybe used; these are available at:http://www2.mrc-lmb.cam.ac.uk/vbase/list2.php.

In a humanised antibody molecule of the present disclosure, the acceptorheavy and light chains do not necessarily need to be derived from thesame antibody and may, if desired, comprise composite chains havingframework regions derived from different chains.

The framework regions need not have exactly the same sequence as thoseof the acceptor antibody. For instance, unusual residues may be changedto more frequently-occurring residues for that acceptor chain class ortype. Alternatively, selected residues in the acceptor framework regionsmay be changed so that they correspond to the residue found at the sameposition in the donor antibody (see Reichmann et al 1998, Nature, 332,323-324). Such changes should be kept to the minimum necessary torecover the affinity of the donor antibody. A protocol for selectingresidues in the acceptor framework regions which may need to be changedis set forth in WO91/09967.

Derivatives of frameworks may have 1, 2, 3 or 4 amino acids replacedwith an alternative amino acid, for example with a donor residue.

Donor residues are residues from the donor antibody, i.e. the antibodyfrom which the CDRs were originally derived, in particular the residuein a corresponding location from the donor sequence is adopted. Donorresidues may be replaced by a suitable residue derived from a humanreceptor framework (acceptor residues).

The residues in antibody variable domains are conventionally numberedaccording to a system devised by Kabat et al. This system is set forthin Kabat et al., 1987, in Sequences of Proteins of ImmunologicalInterest, US Department of Health and Human Services, NIH, USA(hereafter “Kabat et al. (supra)”). This numbering system is used in thepresent specification except where otherwise indicated.

The Kabat residue designations do not always correspond directly withthe linear numbering of the amino acid residues. The actual linear aminoacid sequence may contain fewer or additional amino acids than in thestrict Kabat numbering corresponding to a shortening of, or insertioninto, a structural component, whether framework or complementaritydetermining region (CDR), of the basic variable domain structure. Thecorrect Kabat numbering of residues may be determined for a givenantibody by alignment of residues of homology in the sequence of theantibody with a “standard” Kabat numbered sequence.

The CDRs of the heavy chain variable domain are located at residues31-35 (CDR-H1), residues 50-65 (CDR-H2) and residues 95-102 (CDR-H3)according to the Kabat numbering system. However, according to Chothia(Chothia, C. and Lesk, A. M. J. Mol. Biol., 196, 901-917 (1987)), theloop equivalent to CDR-H1 extends from residue 26 to residue 32. Thusunless indicated otherwise ‘CDR-H1’ as employed herein is intended torefer to residues 26 to 35, as described by a combination of the Kabatnumbering system and Chothia's topological loop definition.

The CDRs of the light chain variable domain are located at residues24-34 (CDR-L1), residues 50-56 (CDR-L2) and residues 89-97 (CDR-L3)according to the Kabat numbering system.

In one example there is provided a binding domain comprising a heavychain variable region (VH), specific for CD79 which comprises threeCDRs, wherein CDR H1 has the sequence given in SEQ ID NO: 78, CDR H2 hasthe sequence given in SEQ ID NO: 79, and CDR H3 has the sequence givenin SEQ ID NO: 80.

In one embodiment there is provided a binding domain comprising a heavychain variable region (VH), specific for CD79 comprising 3 heavy chainCDRs SEQ ID NO: 88 for CDRH1, SEQ ID NO: 89 for CDRH2 and SEQ ID NO: 90for CDRH3.

In one embodiment there is provided the binding domain comprising alight chain variable region specific for CD79 comprising 3 light chainCDRs SEQ ID NO: 75 for CDRL1, SEQ ID NO: 76 for CDRL2 and SEQ ID NO: 77for CDRL3.

In one embodiment there is provided binding domain comprising a lightchain variable region specific for CD79 comprising 3 light chain CDRsSEQ ID NO: 85 for CDRL1, SEQ ID NO: 86 for CDRL2 and SEQ ID NO: 87 forCDRL3.

In one example there is provided a binding domain comprising a heavychain variable region (VH), specific for CD79 which comprises threeCDRs, wherein CDR H1 has the sequence given in SEQ ID NO: 78, CDR H2 hasthe sequence given in SEQ ID NO: 79, and CDR H3 has the sequence givenin SEQ ID NO: 80 and a light chain variable region (VL) which comprisesthree CDRs, wherein CDR L1 has the sequence given in SEQ ID NO: 75, CDRL2 has the sequence given in SEQ ID NO: 76 and CDR L3 has the sequencegiven in SEQ ID NO: 77.

In one example there is provided a binding domain comprising a heavychain variable region (VH), specific for CD79 which comprises threeCDRs, wherein CDR H1 has the sequence given in SEQ ID NO: 88, CDR H2 hasthe sequence given in SEQ ID NO: 89, and CDR H3 has the sequence givenin SEQ ID NO: 90 and a light chain variable region (VL) which comprisesthree CDRs, wherein CDR L1 has the sequence given in SEQ ID NO: 85, CDRL2 has the sequence given in SEQ ID NO: 86 and CDR L3 has the sequencegiven in SEQ ID NO: 87.

In one embodiment a multispecific molecule according to the presentdisclosure comprises a binding domain specific to CD45 which comprises 3heavy chain CDRS selected from the group comprising SEQ ID NO: 98, 99,100, 108, 109, 110, 118, 119, 120, 128, 129 and 130, In one embodiment amultispecific molecule according to the present disclosure comprises abinding domain specific to CD45 which comprises 3 light chain CDRSselected from the group comprising SEQ ID NO: 95, 97, 97, 105, 106, 107,115, 116, 117, 125, 126 and 127.

In one embodiment a multispecific molecule according to the presentdisclosure comprises a binding domain specific to CD45 which comprises 3heavy chain CDRS selected from the group comprising SEQ ID NO: 98, 99,100, 108, 109, 110, 118, 119, 120, 128, 129, 130, and 3 light chain CDRSselected from the group comprising SEQ ID NO: 95, 97, 97, 105, 106, 107,115, 116, 117, 125, 126 and 127.

In one embodiment there is provided a binding domain comprising a heavychain variable region (VH), specific for CD45 comprising 3 heavy chainCDRs SEQ ID NO: 98 for CDRH1, SEQ ID NO: 99 for CDRH2 and SEQ ID NO: 100for CDRH3.

In one embodiment there is provided a binding domain comprising a heavychain variable region (VH), specific for CD45 comprising 3 heavy chainCDRs SEQ ID NO: 108 for CDRH1, SEQ ID NO: 109 for CDRH2 and SEQ ID NO:110 for CDRH3.

In one embodiment there is provided a binding domain comprising a heavychain variable region (VH), specific for CD45 comprising 3 heavy chainCDRs SEQ ID NO: 118 for CDRH1, SEQ ID NO: 119 for CDRH2 and SEQ ID NO:120 for CDRH3.

In one embodiment there is provided a binding domain comprising a heavychain variable region (VH), specific for CD45 comprising 3 heavy chainCDRs SEQ ID NO: 128 for CDRH1, SEQ ID NO: 129 for CDRH2 and SEQ ID NO:130 for CDRH3.

In one embodiment there is provided a binding domain comprising a lightchain variable region specific for CD45 comprising 3 light chain CDRsSEQ ID NO: 95 for CDRL1, SEQ ID NO: 96 for CDRL2 and SEQ ID NO: 97 forCDRL3.

In one embodiment there is provided binding domain comprising a lightchain variable region specific for CD45 comprising 3 light chain CDRsSEQ ID NO: 105 for CDRL1, SEQ ID NO: 106 for CDRL2 and SEQ ID NO: 107for CDRL3.

In one embodiment there is provided binding domain comprising a lightchain variable region specific for CD45 comprising 3 light chain CDRsSEQ ID NO: 115 for CDRL1, SEQ ID NO: 116 for CDRL2 and SEQ ID NO: 117for CDRL3.

In one embodiment there is provided binding domain comprising a lightchain variable region specific for CD45 comprising 3 light chain CDRsSEQ ID NO: 125 for CDRL1, SEQ ID NO: 126 for CDRL2 and SEQ ID NO: 127for CDRL3.

In one example there is provided a binding domain specific to CD45comprising a heavy chain variable region (VH), which comprises threeCDRs, wherein CDR H1 has the sequence given in SEQ ID NO: 98, CDR H2 hasthe sequence given in SEQ ID NO: 99, and CDR H3 has the sequence givenin SEQ ID NO: 100 and a light chain variable region (VL) which comprisesthree CDRs, wherein CDR L1 has the sequence given in SEQ ID NO: 95, CDRL2 has the sequence given in SEQ ID NO: 96 and CDR L3 has the sequencegiven in SEQ ID NO: 97.

In one example there is provided a binding domain specific to CD45comprising a heavy chain variable region (VH), which comprises threeCDRs, wherein CDR H1 has the sequence given in SEQ ID NO: 108, CDR H2has the sequence given in SEQ ID NO: 109, and CDR H3 has the sequencegiven in SEQ ID NO: 110 and a light chain variable region (VL) whichcomprises three CDRs, wherein CDR L1 has the sequence given in SEQ IDNO: 105, CDR L2 has the sequence given in SEQ ID NO: 106 and CDR L3 hasthe sequence given in SEQ ID NO: 107. In one example there is provided abinding domain specific to CD45 comprising a heavy chain variable region(VH), which comprises three CDRs, wherein CDR H1 has the sequence givenin SEQ ID NO: 118, CDR H2 has the sequence given in SEQ ID NO: 119, andCDR H3 has the sequence given in SEQ ID NO: 120 and a light chainvariable region (VL) which comprises three CDRs, wherein CDR L1 has thesequence given in SEQ ID NO: 115, CDR L2 has the sequence given in SEQID NO: 116 and CDR L3 has the sequence given in SEQ ID NO: 117. In oneexample there is provided a binding domain specific to CD45 comprising aheavy chain variable region (VH), which comprises three CDRs, whereinCDR H1 has the sequence given in SEQ ID NO: 128, CDR H2 has the sequencegiven in SEQ ID NO: 129, and CDR H3 has the sequence given in SEQ ID NO:130 and a light chain variable region (VL) which comprises three CDRs,wherein CDR L1 has the sequence given in SEQ ID NO: 125, CDR L2 has thesequence given in SEQ ID NO: 126 and CDR L3 has the sequence given inSEQ ID NO: 127. In one example the present invention provides amultispecific molecule comprising a binding domain specific to theantigen CD79 and a binding domain specific to the antigen CD45 whereinthese pairs of binding domains each comprise 6 CDRs from a pair of CD79and CD45 antibodies, for example selected from the following pairsantibodies; 4447 and 4122, 4447 and 4129, 4447 and 4131, 4447 and 4133,4450 and 4122, 4450 and 4129, 4450 and 4131 and 4447 and 4133.

The sequences of these CD79 antibodies (antibody 4447 and antibody4450), including VH, VL and CDR sequences are provided herein below. Thesequences of these CD45 antibodies (antibodies 4122, 4129, 4131 and4133) including VH, VL and CDR sequences are provided herein below, andmay be combined as binding domains in molecules of the presentinvention. In one embodiment the disclosure extends to an antibodysequence disclosed herein.

In one example there is provided a binding domain specific to albumincomprising a heavy chain variable region (VH), which comprises threeCDRs, wherein CDR H1 has the sequence given in SEQ ID NO: 131, CDR H2has the sequence given in SEQ ID NO: 132, and CDR H3 has the sequencegiven in SEQ ID NO: 133 and a light chain variable region (VL) whichcomprises three CDRs, wherein CDR L1 has the sequence given in SEQ IDNO: 134, CDR L2 has the sequence given in SEQ ID NO: 135 and CDR L3 hasthe sequence given in SEQ ID NO: 136.

In one example there is provided a binding domain specific to albumincomprising a heavy chain variable region (VH) having the sequence givenin SEQ ID NO:137 and a light chain variable region (VL) having thesequence given in SEQ ID NO:139.

In one example there is provided a binding domain specific to albumincomprising a heavy chain variable region (VH) having the sequence givenin SEQ ID NO:138 and a light chain variable region (VL) having thesequence given in SEQ ID NO:140.

In one example the binding domains are humanised.

In one example one or more CDRs provided herein may be modified toremove undesirable residues or sites, such as cysteine residues oraspartic acid (D) isomerisation sites or asparagine (N) deamidationsites. In one example an Asparagine deamidation site may be removed fromone or more CDRs by mutating the asparagine residue (N) and/or aneighbouring residue to any other suitable amino acid. In one example anasparagine deamidation site such as NG or NS may be mutated, for exampleto NA or NT.

In one example an Aspartic acid isomerisation site may be removed fromone or more CDRs by mutating the aspartic acid residue (D) and/or aneighbouring residue to any other suitable amino acid. In one example anaspartic acid isomerisation site such as DG or DS may be mutated, forexample to EG, DA or DT.

For example one or more cysteine residues in any one of the CDRs may besubstituted with another amino acid, such as serine.

In one example an N-glycosylation site such as NLS may be removed bymutating the asparagine residue (N) to any other suitable amino acid,for example to SLS or QLS. In one example an N-glycosylation site suchas NLS may be removed by mutating the serine residue (S) to any otherresidue with the exception of threonine (T).

The skilled person is able to test variants of CDRs or humanisedsequences in any suitable assay such as those described herein toconfirm activity is maintained.

Specific binding to antigen may be tested using any suitable assayincluding for example ELISA or surface plasmon resonance methods such asBIACORE where binding to antigen (CD45 and/or CD79) may be measured.Such assays may use isolated natural or recombinant CD45 or CD79 (a orb) or a suitable fusion protein/polypeptide. In one example binding ismeasured using recombinant CD45 (SEQ ID NO: 143 or amino acids 23-1304of SEQ ID NO:143) or CD79 such as the sequence provided in SEQ ID NO:141and SEQ ID NO:142 and amino acids 33-226 of SEQ ID NO:141 and aminoacids 29-229 of SEQ ID NO:142) by for example surface plasmon resonance,such as BIACORE. Alternatively the proteins may be expressed on a cell,such as a HEK cell and affinity measured employing a flow cytometrybased affinity determination.

The antibody sequences provided by the present invention may be used toidentify further antibodies and hence binding domains suitable for usein the multispecific molecules of the present invention. Antibodieswhich cross-block the binding of an antibody molecule according to thepresent invention to CD79 in particular, an antibody molecule comprisingthe heavy chain sequence given in SEQ ID NO:73 and the light chainsequence given in SEQ ID NO:71 or an antibody molecule comprising theheavy chain sequence given in SEQ ID NO:83 and the light chain sequencegiven in SEQ ID NO:81 may be similarly useful in binding CD79 andtherefore similarly useful in the multispecific molecules of the presentinvention. Accordingly, the present invention also provides amulti-specific molecule comprising a binding domain specific to theantigen CD45 and a binding domain specific to the antigen CD79b whereinthe binding domain for CD79b cross-blocks the binding of any one of theantibody molecules described herein above to CD79 and/or iscross-blocked from binding CD79 by any one of those antibodies. In oneembodiment, such an antibody binds to the same epitope as an antibodydescribed herein above.

Similarly antibodies which cross-block the binding of an antibodymolecule according to the present invention to CD45, in particular, anantibody molecule comprising the heavy chain sequence given in SEQ IDNO:93 and the light chain sequence given in SEQ ID NO:91 or an antibodymolecule comprising the heavy chain sequence given in SEQ ID NO:103 andthe light chain sequence given in SEQ ID NO:101, or an antibody moleculecomprising the heavy chain sequence given in SEQ ID NO:113 and the lightchain sequence given in SEQ ID NO:111, or an antibody moleculecomprising the heavy chain sequence given in SEQ ID NO:123 and the lightchain sequence given in SEQ ID NO:121, or an antibody moleculecomprising the heavy chain sequence given in SEQ ID NO:133 and the lightchain sequence given in SEQ ID NO:131 may be similarly useful in bindingCD45 and therefore similarly useful in the multispecific molecules ofthe present invention. Accordingly, the present invention also providesa multi-specific molecule comprising a binding domain specific to theantigen CD45 and a binding domain specific to the antigen CD79 whereinthe binding domain for CD45 cross-blocks the binding of any one of theantibody molecules described herein above to CD45 and/or iscross-blocked from binding CD45 by any one of those antibodies. In oneembodiment, such an antibody binds to the same epitope as an antibodydescribed herein above. In another embodiment the cross-blockingantibody binds to an epitope which borders and/or overlaps with theepitope bound by an antibody described herein above.

In another embodiment the cross-blocking neutralising antibody binds toan epitope which borders and/or overlaps with the epitope bound by anantibody described herein above. Cross-blocking antibodies can beidentified using any suitable method in the art, for example by usingcompetition ELISA or BIACORE assays where binding of the cross blockingantibody to antigen (CD45 and/or CD79) prevents the binding of anantibody of the present invention or vice versa. Such cross blockingassays may use isolated natural or recombinant CD45 or CD79 (a and/or b)or a suitable fusion protein/polypeptide. In one example binding andcross-blocking is measured using recombinant CD45 (SEQ ID NO: 143) orCD79.

Alternatively or in addition, the antibodies according to this aspect ofthe invention may be cross-blocked from binding to antigen (CD45 orCD79) by an a binding domain disclosed herein, for example comprisingCDRs derived from the the heavy chain variable sequence given in and thelight chain sequence given in SEQ ID NO:71 and 73, 81 and 83, 91 and 93,101 and 103, 111 and 113, and 121 and 123. Also provided therefore is amulti-specific molecule comprising a binding domain specific to theantigen CD45 and a binding domain specific to the antigen CD79b whereinthe binding domain for CD79b cross-blocks the binding of any one of theantibody molecules described herein above to CD79b and/or iscross-blocked from binding CD79 by any one of those antibodies bygreater than 80%, for example by greater than 85%, such as by greaterthan 90%, in particular by greater than 95% and optionally wherein thebinding domain for CD45 cross-blocks the binding of any one of theantibody molecules described herein above to CD45 and/or iscross-blocked from binding CD45 by any one of those antibodies bygreater than 80%, for example by greater than 85%, such as by greaterthan 90%, in particular by greater than 95%.

In one aspect, there is provided a multi-specific antibody moleculecomprising or consisting of:

-   -   a) a polypeptide chain of formula (I):

V_(H)—CH₁—X—(V₁)_(p);

b) a polypeptide chain of formula (II):

V_(L)—C_(L)—Y—(V₂)_(q);

-   -   wherein:    -   V_(H) represents a heavy chain variable domain;    -   CH₁ represents a domain of a heavy chain constant region, for        example domain 1 thereof;    -   X represents a bond or linker, for example an amino acid linker;    -   Y represents a bond or linker, for example an amino acid linker;    -   V₁ represents a dab, scFv, dsscFv or dsFv;    -   V_(L) represents a variable domain, for example a light chain        variable domain;    -   C_(L) represents a domain from a constant region, for example a        light chain constant region domain, such as Ckappa;    -   V₂ represents a dab, scFv, dsscFv or dsFv;    -   p is 0 or 1;    -   q is 0 or 1; and        when p is 1 q is 0 or 1 and when q is 1 p is 0 or 1 i.e. p and q        do not both represent 0

In one embodiment the multispecific antibody molecule comprises no morethan one binding domain for CD45 and no more than one binding domain forCD79

In one embodiment q is 0 and p is 1.

In one embodiment q is 1 and p is 1.

In one embodiment V₁ is a dab and V₂ is a dab and together they form asingle binding domain of a co-operative pair of variable regions, suchas a cognate VH/VL pair, which are optionally linked by a disulphidebond.

In one embodiment V_(H) and V_(L) are specific to, CD79, for exampleCD79a or CD79b.

In one embodiment the V₁ is specific to, CD79, for example CD79a orCD79b.

In one embodiment the V₂ is specific to, CD79, for example CD79a orCD79b.

In one embodiment the V₁ and V₂ together (eg as binding domain) arespecific to, CD79, for example CD79a or CD79b and V_(H) and V_(L) arespecific to, CD45.

In one embodiment the V₁ is specific to, CD45.

In one embodiment the V₂ is specific to, CD45.

In one embodiment the V₁ and V₂ together (eg as one binding domain) arespecific to, CD45 and V_(H) and V_(L) are specific to CD79.

In one embodiment the V₁ is specific to CD45, V₂ is specific to albuminand V_(H) and V_(L) are specific to CD79.

In one embodiment the V₁ is specific to albumin, V₂ is specific to CD45and V_(H) and V_(L) are specific to CD79.

In one embodiment the V₁ is specific to CD79, V₂ is specific to albuminand V_(H) and V_(L) are specific to CD45.

In one embodiment the V₁ is specific to albumin, V₂ is specific to CD79and V_(H) and V_(L) are specific to CD45.

In one embodiment the V₁ is a dsscFv specific to CD45, V₂ is a dsscFvspecific to albumin and V_(H) and V_(L) are specific to CD79.

In one embodiment the V₁ is a dsscFv specific to albumin, V₂ is a dscFvspecific to CD45 and V_(H) and V_(L) are specific to CD79.

In one embodiment the V₁ is a dsscFv specific to CD79, V₂ is a dsscFvspecific to albumin and V_(H) and V_(L) are specific to CD45.

In one embodiment the V₁ is a dsscFv specific to albumin, V₂ is a dsscFvspecific to CD79 and V_(H) and V_(L) are specific to CD45.

V1, V2, VH and VL in the constructs above may each represent a bindingdomain and incorporate any of the sequences provided herein.

X and Y represent any suitable linker, for example X and Y may beSGGGGSGGGGS (SEQ ID NO:17).

In one embodiment, when V₁ and/or V₂ are a dab, dsFv or a dsscFv, thedisulfide bond between the variable domains V_(H) and V_(L) of V₁ and/orV₂ is formed between positions V_(H)44 and V_(L)100.

The present disclosure also extends to novel polypeptide sequencesdisclosed herein and sequences at least 80% similar or identicalthereto, for example 85% or greater, such 90% or greater, in particularby 95% or greater similarity or identity.

“Identity”, as used herein, indicates that at any particular position inthe aligned sequences, the amino acid residue is identical between thesequences. “Similarity”, as used herein, indicates that, at anyparticular position in the aligned sequences, the amino acid residue isof a similar type between the sequences. For example, leucine may besubstituted for isoleucine or valine.

Other amino acids which can often be substituted for one another includebut are not limited to:

-   -   phenylalanine, tyrosine and tryptophan (amino acids having        aromatic side chains);    -   lysine, arginine and histidine (amino acids having basic side        chains);    -   aspartate and glutamate (amino acids having acidic side chains);    -   asparagine and glutamine (amino acids having amide side chains);        and    -   cysteine and methionine (amino acids having sulphur-containing        side chains).

Degrees of identity and similarity can be readily calculated(Computational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing. Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis ofSequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey, 1994; Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press, 1987, Sequence Analysis Primer, Gribskov, M.and Devereux, J., eds., M Stockton Press, New York, 1991, the BLASTsoftware available from NCBI (Altschul, S. F. et al., 1990, J. Mol.Biol. 215:403-410; Gish, W. & States, D. J. 1993, Nature Genet.3:266-272. Madden, T. L. et al., 1996, Meth. Enzymol. 266:131-141;Altschul, S. F. et al., 1997, Nucleic Acids Res. 25:3389-3402; Zhang, J.& Madden, T. L. 1997, Genome Res. 7:649-656,).

In particular in one aspect the present inventon provides the CD45 andCD79 antibodies described herein in any suitable antibody format.

Accordingly in one aspect the present invention provides anti-CD45antibodies or fragments thereof containing one or more of the bindingdomains described herein above comprising the CDRs provided herein andin SEQ ID NOS 95, 96, 97, 98, 99 and 100 (antibody 4122) or 105, 106,107, 108, 109 and 110 (antibody 4129) or 115, 116, 117, 118, 119 and 120(antibody 4131) or 125, 126, 127, 128, 129 and 130 (antibody 4133). Alsoprovided are anti-CD79 antibodies or fragments thereof containing one ormore of the binding domains described herein above comprising the CDRsprovided herein and in SEQ ID NOS 75, 76, 77, 78, 79 and 80 (antibody4447) or SEQ ID NOs 85, 86, 87, 88, 89 and 90 (antibody 4450).

Said CDRs may be incorporated into any suitable antibody framework andinto any suitable antibody format. Such antibodies include wholeantibodies and functionally active fragments or derivatives thereofwhich may be, but are not limited to, monoclonal, humanised, fully humanor chimeric antibodies. Accordingly, such antibodies may comprise acomplete antibody molecule having full length heavy and light chains ora fragment thereof and may be, but are not limited to Fab, modified Fab,Fab′, F(ab′)₂, Fv, single domain antibodies, scFv, bi, tri ortetra-valent antibodies, Bis-scFv, diabodies, triabodies, tetrabodiesand epitope-binding fragments of any of the above (see for exampleHolliger and Hudson, 2005, Nature Biotech. 23(9):1126-1136; Adair andLawson, 2005, Drug Design Reviews—Online 2(3), 209-217). The methods forcreating and manufacturing these antibody fragments are well known inthe art (see for example Verma et al., 1998, Journal of ImmunologicalMethods, 216, 165-181). Multi-valent antibodies may comprise multiplespecificities or may be monospecific (see for example WO 92/22853 andWO05/113605). It will be appreciated that this aspect of the inventionalso extends to variants of these anti-CD45 and CD79 antibodiesincluding humanised versions and modified versions, including those inwhich amino acids have been mutated in the CDRs to remove one or moreisomerisation, deamidation, glycosylation site or cysteine residue asdescribed herein above.

Linkers

The teaching herein of linkers in one context can equally be applied tolinkers in different contexts where a linker is employed, such as in anymultispecific molecule of the present invention.

In one embodiment, the linker employed in a molecule of the disclosureis an amino acid linker 50 residues or less in length, for exampleselected from a sequence shown in sequence 5 to 70.

TABLE 1 Hinge linker sequences SEQ ID NO: SEQUENCE  5 DKTHTCAA  6DKTHTCPPCPA  7 DKTHTCPPCPATCPPCPA  8 DKTHTCPPCPATCPPCPATCPPCPA  9DKTHTCPPCPAGKPTLYNSLVMSDTAGTCY 10 DKTHTCPPCPAGKPTHVNVSVVMAEVDGTCY 11DKTHTCCVECPPCPA 12 DKTHTCPRCPEPKSCDTPPPCPRCPA 13 DKTHTCPSCPA

TABLE 2 Flexible linker sequences SEQ ID NO: SEQUENCE 14 SGGGGSE 15DKTHTS 16 (S)GGGGS 17 (S)GGGGSGGGGS 18 (S)GGGGSGGGGSGGGGS 19(S)GGGGSGGGGSGGGGSGGGGS 20 (S)GGGGSGGGGSGGGGSGGGGSGGGGS 21 AAAGSG-GASAS22 AAAGSG-XGGGS-GASAS 23 AAAGSG-XGGGSXGGGS-GASAS 24AAAGSG-XGGGSXGGGSXGGGS-GASAS 25 AAAGSG-XGGGSXGGGSXGGGSXGGGS-GASAS 26AAAGSG-XS-GASAS 27 PGGNRGTTTTRRPATTTGSSPGPTQSHY 28 ATTTGSSPGPT 29 ATTTGS30 GS 31 EPSGPISTINSPPSKESHKSP 32 GTVAAPSVFIFPPSD 33 GGGGIAPSMVGGGGS 34GGGGKVEGAGGGGGS 35 GGGGSMKSHDGGGGS 36 GGGGNLITIVGGGGS 37 GGGGVVPSLPGGGGS38 GGEKSIPGGGGS 39 RPLSYRPPFPFGFPSVRP 40 YPRSIYIRRRHPSPSLTT 41TPSHLSHILPSFGLPTFN 42 RPVSPFTFPRLSNSWLPA 43 SPAAHFPRSIPRPGPIRT 44APGPSAPSHRSLPSRAFG 45 PRNSIHFLHPLLVAPLGA 46 MPSLSGVLQVRYLSPPDL 47SPQYPSPLTLTLPPHPSL 48 NPSLNPPSYLHRAPSRIS 49 LPWRTSLLPSLPLRRRP 50PPLFAKGPVGLLSRSFPP 51 VPPAPVVSLRSAHARPPY 52 LRPTPPRVRSYTCCPTP- 53PNVAHVLPLLTVPWDNLR 54 CNPLLPLCARSPAVRTFP

(S) is optional in sequences 17 to 20.

Examples of rigid linkers include the peptide sequences GAPAPAAPAPA (SEQID NO:69), PPPP (SEQ ID NO:70) and PPP.

Other linkers are shown in Table 3:

SEQ ID NO: SEQUENCE 55 DLCLRDWGCLW 56 DICLPRWGCLW 57 MEDICLPRWGCLWGD 58QRLMEDICLPRWGCLWEDDE 59 QGLIGDICLPRWGCLWGRSV 60 QGLIGDICLPRWGCLWGRSVK 61EDICLPRWGCLWEDD 62 RLMEDICLPRWGCLWEDD 63 MEDICLPRWGCLWEDD 64MEDICLPRWGCLWED 65 RLMEDICLARWGCLWEDD 66 EVRSFCTRWPAEKSCKPLRG 67RAPESFVCYWETICFERSEQ 68 EMCYFPGICWM

Effector Molecules

If desired a multispecific molecule for use in the present invention maybe conjugated to one or more effector molecule(s). It will beappreciated that the effector molecule may comprise a single effectormolecule or two or more such molecules so linked as to form a singlemoiety that can be attached to the multispecific molecules of thepresent invention. Where it is desired to obtain an antibody ormultispecific molecule according to the present disclosure linked to aneffector molecule, this may be prepared by standard chemical orrecombinant DNA procedures in which the antibody fragment is linkedeither directly or via a coupling agent to the effector molecule.Techniques for conjugating such effector molecules to antibodies arewell known in the art (see, Hellstrom et al., Controlled Drug Delivery,2nd Ed., Robinson et al., eds., 1987, pp. 623-53; Thorpe et al., 1982,Immunol. Rev., 62:119-58 and Dubowchik et al., 1999, Pharmacology andTherapeutics, 83, 67-123). Particular chemical procedures include, forexample, those described in WO 93/06231, WO 92/22583, WO 89/00195, WO89/01476 and WO 03/031581. Alternatively, where the effector molecule isa protein or polypeptide the linkage may be achieved using recombinantDNA procedures, for example as described in WO 86/01533 and EP0392745.

In one embodiment the multispecific molecules of the present disclosuremay comprise an effector molecule.

The term effector molecule as used herein includes, for example,antineoplastic agents, drugs, toxins, biologically active proteins, forexample enzymes, other antibody or antibody fragments, synthetic ornaturally occurring polymers, nucleic acids and fragments thereof e.g.DNA, RNA and fragments thereof, radionuclides, particularly radioiodide,radioisotopes, chelated metals, nanoparticles and reporter groups suchas fluorescent compounds or compounds which may be detected by NMR orESR spectroscopy.

Examples of effector molecules may include cytotoxins or cytotoxicagents including any agent that is detrimental to (e.g. kills) cells.Examples include combrestatins, dolastatins, epothilones, staurosporin,maytansinoids, spongistatins, rhizoxin, halichondrins, roridins,hemiasterlins, taxol, cytochalasin B, gramicidin D, ethidium bromide,emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine,colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione,mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol, andpuromycin and analogs or homologs thereof.

Effector molecules also include, but are not limited to, antimetabolites(e.g. methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g. mechlorethamine,thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g. daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g. dactinomycin (formerly actinomycin),bleomycin, mithramycin, anthramycin (AMC), calicheamicins orduocarmycins), and anti-mitotic agents (e.g. vincristine andvinblastine).

Other effector molecules may include chelated radionuclides such as¹¹¹In and ⁹⁰Y, Lu¹⁷⁷, Bismuth²¹³, Californium²⁵², Iridium¹⁹² andTungsten¹⁸⁸/Rhenium¹⁸⁸; or drugs such as but not limited to,alkylphosphocholines, topoisomerase I inhibitors, taxoids and suramin.

Other effector molecules include proteins, peptides and enzymes. Enzymesof interest include, but are not limited to, proteolytic enzymes,hydrolases, lyases, isomerases, transferases. Proteins, polypeptides andpeptides of interest include, but are not limited to, immunoglobulins,toxins such as abrin, ricin A, pseudomonas exotoxin, or diphtheriatoxin, a protein such as insulin, tumour necrosis factor, α-interferon,β-interferon, nerve growth factor, platelet derived growth factor ortissue plasminogen activator, a thrombotic agent or an anti-angiogenicagent, e.g. angiostatin or endostatin, or, a biological responsemodifier such as a lymphokine, interleukin-1 (IL-1), interleukin-2(IL-2), granulocyte macrophage colony stimulating factor (GM-CSF),granulocyte colony stimulating factor (G-CSF), nerve growth factor (NGF)or other growth factor and immunoglobulins.

Other effector molecules may include detectable substances useful forexample in diagnosis. Examples of detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, radioactive nuclides, positronemitting metals (for use in positron emission tomography), andnonradioactive paramagnetic metal ions. See generally U.S. Pat. No.4,741,900 for metal ions which can be conjugated to antibodies for useas diagnostics. Suitable enzymes include horseradish peroxidase,alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;suitable prosthetic groups include streptavidin, avidin and biotin;suitable fluorescent materials include umbelliferone, fluorescein,fluorescein isothiocyanate, rhodamine, dichlorotriazinylaminefluorescein, dansyl chloride and phycoerythrin; suitable luminescentmaterials include luminol; suitable bioluminescent materials includeluciferase, luciferin, and aequorin; and suitable radioactive nuclidesinclude ¹²⁵I, ¹³¹I, ¹¹¹In and ⁹⁹Tc.

In another example the effector molecule may increase the half-life ofthe antibody in vivo, and/or reduce immunogenicity of the antibodyand/or enhance the delivery of an antibody across an epithelial barrierto the immune system. Examples of suitable effector molecules of thistype include polymers, albumin, albumin binding proteins or albuminbinding compounds such as those described in WO05/117984.

Where the effector molecule is a polymer it may, in general, be asynthetic or a naturally occurring polymer, for example an optionallysubstituted straight or branched chain polyalkylene, polyalkenylene orpolyoxyalkylene polymer or a branched or unbranched polysaccharide, e.g.a homo- or hetero-polysaccharide.

Specific optional substituents which may be present on theabove-mentioned synthetic polymers include one or more hydroxy, methylor methoxy groups.

Specific examples of synthetic polymers include optionally substitutedstraight or branched chain poly(ethyleneglycol), poly(propyleneglycol)poly(vinylalcohol) or derivatives thereof, especially optionallysubstituted poly(ethyleneglycol) such as methoxypoly(ethyleneglycol) orderivatives thereof.

Functional Assays and Screening Formats

Typically suitable binding domains for use in the present invention canbe identified by testing one or more binding domain pairs in afunctional assay. For example a multi specific molecule comprising abinding domain specific to the antigen CD45 and a binding domainspecific to the antigen CD79a and/or CD79b may be tested in one or morefunctional assays.

A “functional assay,” as used herein, is an assay that can be used todetermine one or more desired properties or activities of the proteincomplexes, antibody complexes or the mixture of antibodies subject tothe assay conditions. Suitable functional assays may be binding assays,apoptosis assays, antibody-dependent cellular cytotoxicity (ADCC)assays, complement-dependent cytotoxicity (CDC) assays, inhibition ofcell growth or proliferation (cytostatic effect) assays, cell-killing(cytotoxic effect) assays, cell-signaling assays, cytokine productionassays, antibody production and isotype switching, and cellulardifferentiation assays.

The efficacy of multispecific antibodies according to the presentdisclosure can be compared to individual antibodies or mixtures ofantibodies (or fragments) in such models by methods generally known toone of ordinary skill in the art.

The functional assays may be repeated a number of times as necessary toenhance the reliability of the results. Various statistical tests knownto the skilled person can be employed to identify statisticallysignificant results and thus identify multispecific molecules withbiological functions.

Examples of suitable functional assays are described in the Examplesherein and include measuring the ability of a multispecific molecule ofthe present invention to inhibit B cell activation following stimulationwith anti-IgM, as measured by detecting the inhibition of markers of Bcell activation such as phosphorylated Akt expression, phosphorylatedP38 expression, PLCγ signalling, CD40 expression, CD71 expression and/orCD86 expression.

When establishing a functional assay for screening the skilled personcan set a suitable threshold over which an identified activity is deemeda ‘hit’. Where more than one functional assay is used the threshold foreach assay may be set at a suitable level to establish a manageable hitrate, in one example the hit rate may be 3-5%, in one example thecriteria set when searching for pairs of binding domains that inhibit Bcell function may be at least 30% inhibition of at least twophospho-readouts in a B cell activation assay.

In one example a multispecific molecule of the present invention has anIC50 of less than 5 nM for inhibition of CD86 expression in anti-IgMstimulated B cells.

In one embodiment in vivo assays, such as animal models, including mousetumor models, models of auto-immune disease, virus-infected orbacteria-infected rodent or primate models, and the like, may beemployed to test molecules of the present disclosure.

An example of a suitable format for screening and discovery of bindingdomains is described herein below.

Screening to identify binding domains for use in the present inventionmay employ a bispecific protein complex.

“Bispecific protein complex” as used herein refers to a moleculecomprising two proteins (A and B referred to herein as bispecificcomponents) which are retained together by a heterodimeric-tether. Inone embodiment one or both of the proteins have a binding domain, forexample one or both of the proteins are antibodies or fragments thereof.

Typically the bispecific protein complex has the formula A-X:Y-Bwherein:

-   -   A-X is a first fusion protein;    -   Y-B is a second fusion protein;    -   X:Y is a heterodimeric-tether;    -   A comprises a first binding domain;    -   B comprises a second binding domain;    -   X is a first binding partner of a binding pair;    -   Y is a second binding partner of the binding pair; and    -   : is an interaction (such as a binding interaction) between X        and Y.

“Fusion proteins” as employed herein comprise a protein component, forexample A or B fused to another entity, for example a binding partner Xor Y (as appropriate). In embodiment the fusion protein is atranslational protein expressed by a recombinant techniques from agenetic construct, for example expressed in a host from a DNA construct.

The function of the tether X:Y is to retain the proteins A and B inproximity to each other so that synergistic function of A and B can berealised.

“heterodimeric-tether” as used herein refers to a tether comprising twodifferent binding partners X and Y which form a interaction (such as abinding) between each other which has an overall affinity that issufficient to retain the two binding partners together. In oneembodiment X and/or Y are unsuitable for forming homodimers.

Heterodimerically-tethered and heterodimeric-tether are usedinterchangeably herein.

In one embodiment “unsuitable for forming homodimers” as employed hereinrefers to formation of the heterodimers of X-Y are more preferable, forexample stable, such as thermodynamically stable and/or physicallystable (for example evidenced by lack of aggregation), once formed.

In one embodiment the X-Y interaction is more favourable than the X-X orY-Y interaction. This reduces the formation of homodimers X-X or Y-Ywhen the fusion proteins A-X and B-Y are mixed. This also rendersremoval of homodimers relatively simple, for example, one purificationstep, such as column chromatography provides substantially pure fusionproteins and/or bispecific protein complexes according to the presentdisclosure.

In one embodiment a purification step is provided after expression ofthe fusion protein. Thus in one embodiment prior to in vitro mixing thefusion protein(s) is/are provided in substantially pure form.Substantially pure form as employed herein refers to wherein the fusionprotein is in the range 85 to 100%, for example 90, 91, 92, 93, 94, 95,96, 97, 98, 99 or 100% monomer. In one embodiment no purification isrequired after the bispecific protein complex formation. In oneembodiment the ratio of fusion proteins employed in the in vitro mixingstep of the present method is A-X to B-Y 0.8:1 to 3:1, such as 1.5:1 or2:1.

In one embodiment the ratio of fusion proteins employed in the in vitromixing step of the present method is B-Y to A-X 0.8:1 to 3:1, such as1.5:1 or 2:1.

In one embodiment the ratio is 1:1.

In one embodiment one (or at least one) of the binding partners isincapable of forming a homodimer, for example an amino acid sequence ofthe binding partner is mutated to eliminate or minimise the formation ofhomodimers.

In one embodiment both of the binding partners are incapable of forminga homodimer, for example one of the binding partners is a peptide andthe other binding partner is a V_(HH) specific to said peptide.

In one embodiment an scFv employed in the molecules of the presentdisclosure is incapable of forming a homodimer.

Incapable of forming homodimers as employed herein, refers to a low orzero propensity to form homodimers. Low as employed herein refers to 5%or less, such as 4, 3, 2, 1, 0.5% or less aggregate.

Small amounts of aggregate in the fusion proteins or residual in theheterodimerically-tethered bispecific protein complex generally hasminimal effect on the method of the present disclosure.

In one embodiment: is a binding interaction, for example based onattractive forces such as Van der Waals forces, such as hydrogen bondingand electrostatic interactions, for example, based on antibodyspecificity for an antigen, such as a peptide.

In one embodiment: is a covalent bond formed from a specific chemicalinteraction, such as click chemistry.

In one embodiment: is not a covalent bond.

“Form the complex” as employed herein refers to an interaction,including a binding interactions or a chemical reaction, which issufficiently specific and strong when the fusion protein components A-Xand B-Y are brought into contact under appropriate conditions that thecomplex is assembled and the fusion proteins are retained together.

“Retained together” as employed herein refers to the holding of thecomponents (the fusion proteins) in the proximity of each other, suchthat after binding the complex can be handled as if it were onemolecule, and in many instances behaves and acts like a single molecule.In one embodiment the retention renders the complex suitable for use inthe method disclosed herein, i.e. suitable for use in at least onefunctional screen.

In one embodiment the binding interaction is reversible.

Specificity when in relation to X and Y as employed herein refers wherethe binding partners X and Y in the interaction only recognise eachother or have significantly higher affinity for each other in comparisonto non-partners, for example at least 2, 3, 4, 5, 6, 7, 8, 9, 10 timeshigher affinity.

In one embodiment, the binding interaction between X and Y has a lowdissociation constant. Examples of a low dissociation constant include1-9×10⁻²s⁻¹ or less, for example 1-9×10⁻³s⁻¹, 1-9×10⁻⁴s⁻¹, 1-9×10⁻⁵s⁻¹,1-9×10⁻⁶s⁻¹ or 1-9×10⁻⁷s⁻¹. Particularly suitable dissociation constantsinclude 1×10⁻⁴s⁻¹ or less, for example 1×10⁻⁵s⁻¹, 1×10⁻⁶s⁻¹ or1×10⁻⁷s⁻¹.

Whilst not wishing to be bound by theory it is thought that the lowdissociation constant (also referred to as off rate) allows themolecules to be sufficiently stable to render the bispecific proteincomplex useful, in particular in functional screening assays.

In one embodiment, the affinity of X and Y for each other is 5 nM orstronger, for example 4 nM, 3 nM, 2 nM, 1 nM or stronger.

In one embodiment, the affinity of X and Y for each other is 900 pM orstronger, such as 800, 700, 600, 500, 400, 300, 200, 100 or 50 pM orstronger.

In another embodiment, the affinity of X and Y for each other is 10 pMor stronger, for example 9, 8, 7, 6 or 5 pM.

Affinity is a value calculated from the on and off rate of aninteraction. The term “affinity” as used herein refers to the strengthof the sum total of non-covalent interactions between a single bindingsite of a molecule (e.g. an antibody) and its binding partner (e.g. apeptide). The affinity of a molecule for its binding partner cangenerally be represented by the equilibrium dissociation constant(K_(D)). Affinity can be measured by common methods known in the art,including those described herein, such as surface plasmon resonancemethods, in particular BIACORE.

In one embodiment, multiple bispecific protein complexes according tothe present disclosure are tested in parallel or essentiallysimultaneously.

Simultaneously as employed herein refers to the where thesamples/molecules/complexes are analysed in the same analysis, forexample in the same “run”.

In one embodiment simultaneously refers to concomitant analysis wherethe signal output is analysed by the instrument at essentially the sametime. This signal may require deconvolution to interpret the resultsobtained.

Advantageously, testing multiple bispecific protein complexes allows formore efficient screening of a large number of bispecific proteincomplexes and the identification of new and interesting relationships.Clearly different variable regions to the target antigens of interestingCD45 and CD79 can give access to subtle nuances in biological function.

In one embodiment, the multiple bispecific protein complexes are testedby using a multiplex as defined above and subjecting the same to one ormore functional assays.

The term “biological function” as used herein refers to an activity thatis natural to or the purpose of the biological entity being tested, forexample a natural activity of a cell, protein or similar. Ideally thepresence of the function can be tested using an in vitro functionalassay, including assays utilizing living mammalian cells. Naturalfunction as employed herein includes aberrant function, such asfunctions associated with cancers.

A relevant “biological comparator” as employed herein refers to asuitable entity for assessing activity, in the same assay as thatemployed for the bispecific protein complex, to establish if there isany change or novel activity or function. Suitable comparators forA-X:Y-B may include purified protein (including recombinant proteins) ina natural form or presented in the same format as the bispecific i.e.where A and B are the same entity, such as A-X:Y-A or B-X:Y-B.Alternatively the fusion protein A-X or B-Y in an uncomplexed form maybe employed as a comparator. Alternatively, multiple comparators ofdifferent formats (in particular as described herein) may be employed.The person skilled in the art is able to identify and include a suitablecontrol/comparator based on common general knowledge or information thatis found in the literature.

The term “synergistic function” as used herein refers to biologicalactivity that is not observed or higher than observed when the first andsecond proteins of a bispecific protein complex of the presentdisclosure are not employed together, for example activity which is onlyobserved in a bispecific form. Therefore, “synergistic” includes novelbiological function.

The present disclosure provides a molecule with at least specificity toCD45 and CD79 with a novel biological function.

Novel biological function as employed herein refers to function which isnot apparent or absent until the two or more synergistic entities[protein A and protein B] are brought together (as a bispecific orotherwise) or a previously unidentified function.

Higher as employed herein refers to an increase in activity including anincrease from zero i.e. some activity in the bispecific where theindividual uncomplexed bispecific component or components has/have noactivity in the relevant functional assay, also referred to herein asnew activity or novel biological function. Higher as employed hereinalso includes a greater than additive function in the bispecific in arelevant functional assay in comparison to the individual uncomplexedbispecific components or bivalent binding domains, for example 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 200, 300% or more increase in arelevant activity.

In one embodiment the novel synergistic function is a higher inhibitoryactivity.

In one embodiment the multispecific antibody molecule of the presentinvention has a higher inhibitory activity than the sum of the activityof a bivalent binding domain to CD45 and a bivalent binding domain toCD79a provided alone or in admixture

In one embodiment, at least one of the first binding partner, X, and thesecond binding partner, Y, of the binding pair are independentlyselected from a peptide and a protein; for example the first bindingpartner or second binding partner is a peptide.

Suitable peptides include the group comprising GCN4, Fos/Jun (human andmurine Fos have a Uniprot number P01100 and P01101 respectively andhuman and murine jun have a Uniprot number P05412 and P05627respectively), human influenza hemagglutinin (HA), polyhistidine (His),green fluorescent protein (GFP) and FLAG. Other peptides are alsocontemplated as suitable for use in the present disclosure andparticularly suitable peptides are affinity tags for proteinpurification because such peptides have a tendency to bind with highaffinity to their respective binding partners.

The term “peptide” as used herein refers to a short polymer of aminoacids linked by peptide bonds, wherein the peptide contains in the rangeof 2 to 100 amino acids, for example 5 to 99, such as 6 to 98, 7 to 97or 8 to 96. In one embodiment a peptide employed in the presentdisclosure is an amino acid sequence of 50 amino acid residues or less,for example 40, 30, 10 or less. The peptides used in the presentdisclosure are of a sufficient length to be fit for purpose, for exampleif the peptide is a linker, it needs to be suitably long to allow thefragment which it links to perform its biological function;alternatively if the peptide is a binding partner, it must be capable ofbinding specifically to another entity such as an antibody. In oneembodiment, the other binding partner of the binding pair (thealternative first or second binding partner) is a protein.

Protein as employed herein refers to an amino acid sequence of 100 aminoacids or more. In one embodiment a “protein” as employed herein refersto an amino acid sequence with a secondary or tertiary structure.

In one embodiment, the first protein, A, and/or second protein, B, ofthe bispecific protein complex is an antibody or antibody fragment. Sucha bispecific protein complex may be referred to as a bispecific antibodycomplex.

In one embodiment each antibody or fragment employed in the bispecificantibody complex of the disclosure comprises one binding site.

The full length antibody or antibody fragment employed in the fusionproteins (A-X or B-Y) may be monospecific, multivalent or bispecific.

Advantageously, the use of two bispecific antibody or antibody fragmentsallows the molecules of the present disclosure, such as the bispecificantibody complex described herein to potentially be specific for up to 4different antigens (i.e. the complex may be tetraspecific). This allowsavidity type effects to be investigated.

In one embodiment, the antibody or antibody fragment employed in themolecules of the present disclosure or components thereof, such as thefirst fusion protein A-X is a monospecific antibody or antibodyfragment, for example a Fab, Fab′, scFv or similar, and in particular isspecific to CD45.

In one embodiment, the antibody or antibody fragment employed in themolecules of the present disclosure or components thereof, such as thesecond fusion protein B-Y is a monospecific antibody or antibodyfragment, for example a Fab, Fab′, scFv or similar, and in particular isspecific to CD79a and/or CD79b.

In one embodiment, the antibody or antibody fragment employed in themolecules of the present disclosure or components thereof, such as thesecond fusion protein B-Y is multivalent, that is has two or morebinding domains.

In one embodiment, the antibody or antibody fragment employed in themolecules of the present disclosure or components thereof, such as thefirst fusion protein A-X is monovalent and the antibody or antibodyfragment employed in the molecules of the present disclosure orcomponents thereof, such as the second fusion protein B-X is monovalent.

Thus in one embodiment the binding domains of the multispecificmolecules of the present disclosure are monovalent.

Thus in one embodiment the binding domains of the multispecificmolecules of the present disclosure are monovalent and monospecific.

In one embodiment, the antibody or antibody fragment employed in themolecules of the present disclosure or components thereof, such as thefirst fusion protein A-X is monovalent and the antibody or antibodyfragment employed in the molecules of the present disclosure orcomponents thereof, such as the second fusion protein B-Y ismultivalent.

In one embodiment, the antibody or antibody fragment employed in themolecules of the present disclosure or components thereof, such as thefirst fusion protein A-X is multivalent and the antibody or antibodyfragment employed in the molecules of the present disclosure orcomponents thereof, such as the second fusion protein B-Y is monovalent.

In one embodiment, the antibody or antibody fragment employed in themolecules of the present disclosure or components thereof, such as thefirst fusion protein A-X is multivalent and the antibody or antibodyfragment employed in the molecules of the present disclosure orcomponents thereof, such as the second fusion protein B-Y ismultivalent.

In one embodiment, a first antibody, a second antibody or both the firstand second antibody of a the molecules of the present disclosure orcomponents thereof, such as a bispecific antibody complex may be an IgGformat, for example an anti-CD45 and/or anti-CD79 antibody may beprovided in an IgG format.

In one embodiment, an antibody fragment is selected from the groupconsisting of: a fragment antigen (Fab) fragment, a single chainvariable fragment (scFv) and a single domain antibody (sdAb), such as ascFv, is employed in the first (A-X) or second fusion protein (B-Y).Advantageously, the small size of a scFv may facilitate the correctfolding of the bispecific antibody complexes.

In one embodiment, the first (A), second antibody/fragment (B) or boththe first and second antibody/fragment of the bispecific antibodycomplex of the present disclosure may be a Fab. In one embodiment, thefirst, second antibody/fragment or both the first and secondantibody/fragment of the bispecific antibody complex of the presentdisclosure is/are a Vim. “Fusion protein” as employed in the context ofa bispecific complex of the present disclosure refers to a protein, forexample an antibody or antibody fragment attached to a binding partner.For convenience bispecific protein complexes of the present disclosureare referred to herein as A-X:Y-B. However, this nomenclature is notintended to limit how the fusion protein A-X and B-Y are designedbecause our experiments indicate that binding partners X and Y can bereversed i.e. A-Y and B-X without adversely impacting on the method.Thus A and B and X and Y are nominal labels referred to for assistingthe explanation of the present technology. “Attached” as employed hereinrefers to connected or joined directly or indirectly via a linker, suchas a peptide linker examples of which are discussed below. Directlyconnected includes fused together (for example a peptide bond) orconjugated chemically.

“Binding partner” as employed herein refers to one component part of abinding pair.

In one embodiment, the affinity of the binding partners is high, 5 nM orstronger, such as 900, 800, 700, 600, 500, 400, 300 pM or stronger.

“Binding pair” as employed herein refers to two binding partners whichspecifically bind to each other. Examples of a binding pair include apeptide and an antibody or binding fragment specific thereto, or anenzyme and ligand, or an enzyme and an inhibitor of that enzyme.

In one embodiment, the first binding partner (X) is selected from thegroup comprising: a full length antibody, a Fab, a Fab′, a scFv, apeptide and a sdAb, wherein examples of a sdAb include VH or VL orV_(H)H.

In one embodiment, the second partner (Y) is selected from the groupcomprising: a full length antibody, a Fab, a Fab′, a scFv, a peptide anda sdAb, wherein examples of a sdAb include VH or VL or V_(H)H.

In one embodiment, where A is an antibody or fragment thereof the firstbinding partner (X) is attached to the C-terminal of the heavy or lightchain of the first antibody or antibody fragment, for example, the firstbinding partner is attached to the C-terminal of the heavy chain of thefirst antibody or antibody fragment.

In another embodiment, where B is an antibody or fragment thereof thesecond binding partner (Y) is attached to the C-terminal of the heavy orlight chain of the second antibody or antibody fragment, for example thesecond binding partner is attached to the C-terminal of the heavy chainof the second antibody or antibody fragment.

In one embodiment X is attached to the C-terminal of the heavy chain ofthe antibody or fragment (protein A) and Y is attached to the C-terminalof the antibody or fragment (protein B).

In one embodiment X is attached via a linker (such as ASGGGG orASGGGGSG) to the C-terminal of the heavy chain of the antibody orfragment (protein A) and Y is attached via a linker (such as ASGGGG orASGGGGSG) to the C-terminal of the antibody or fragment (protein B).

In one embodiment, the first or second binding partner (X or Y) is apeptide.

Examples of a suitable binding pair may include GCN4 (SEQ ID NO: 1) or avariant thereof and 52SR4 (SEQ ID NO:3) or a variant thereof, which is ascFv specific for GCN4.

In a one embodiment, the first binding partner (nominally X) is GCN4(for example as shown in SEQ ID NO:1) or a variant thereof (for examplewithout the His tag) and the second binding partner (nominally Y) is ascFv specific for GCN4 (for example as shown in SEQ ID NO:3) or avariant thereof.

In a one embodiment, the first binding partner (nominally X) is a sFvspecific for GCN4 (for example as shown in SEQ ID NO:3) or a variantthereof and the second binding partner (nominally Y) is GCN4 (forexample as shown in SEQ ID NO:1) or a variant thereof. GCN4 variantsinclude an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%,93%, 94% 95%, 96%, 97% or 98%, or 99% identity to SEQ ID NO:1. GCN4variants also include an amino acid having at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% to a sequence encoded by anucleotide sequence SEQ ID NO:2, or a nucleotide sequence whichhybridises to SEQ ID NO: 2 under stringent conditions.

A suitable scFv specific to GCN4 is 52SR4 (SEQ ID NO: 3) or a variantthereof. Variants of 52SR4 include an amino acid sequence with at least80%, or 85%, or 90%, or 95%, or 98%, or 99% identity to SEQ ID NO: 3.52SR4 variants also include an amino acid sequence having at least atleast 80%, or 85%, or 90%, or 95%, or 98%, or 99% to a sequence encodedby a nucleotide sequence SEQ ID NO:4, or a nucleotide sequence whichhybridises to SEQ ID NO: 2 under stringent conditions.

The present inventors have found that the single chain antibody 52SR4and peptide GCN4, are a binding pair suitable for use in the bispecificprotein complexes of the present disclosure. Alternatively, any suitableantibody/fragment and antigen (such as a peptide) may be employed as Xand Y.

In one embodiment, the first binding partner (X) and the second bindingpartner (Y) are a protein.

In one embodiment, the first binding partner (X) is an enzyme or anactive fragment thereof and the second binding partner (Y) is a ligandor vice versa.

In one embodiment, the first binding partner (X) is an enzyme or anactive fragment thereof and the second binding partner (Y) is aninhibitor of that enzyme or vice versa.

“Active fragment” as employed herein refers to an amino acid fragment,which is less than the whole amino acid sequence for the entity andretains essentially the same biological activity or a relevantbiological activity, for example greater than 50% activity such as 60%,70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

In another embodiment, the first binding partner X is glutathione (GSH)and the second binding partner Y is glutathione-S-transferase (GST) orvice versa.

In another embodiment, X is Fos and Y is Jun or vice versa.

In another embodiment, X is His and Y is anti-His or vice versa.

In another embodiment, the binding pair is clamodulin binding peptideand Y is calmodulin or vice versa.

In another embodiment, X is maltose-binding protein and Y is ananti-maltose binding protein or fragment thereof or vice versa.

Other enzyme-ligand combinations are also contemplated for use inbinding partners. Also suitable are affinity tags known in the art forprotein purification because these have a tendency to bind with highaffinity to their respective binding partners.

Degrees of identity and similarity can be readily calculated(Computational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing. Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis ofSequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey, 1994; Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press, 1987, Sequence Analysis Primer, Gribskov, M.and Devereux, J., eds., M Stockton Press, New York, 1991, the BLASTsoftware available from NCBI (Altschul, S. F. et al., 1990, J. Mol.Biol. 215:403-410; Gish, W. & States, D. J. 1993, Nature Genet.3:266-272. Madden, T. L. et al., 1996, Meth. Enzymol. 266:131-141;Altschul, S. F. et al., 1997, Nucleic Acids Res. 25:3389-3402; Zhang, J.& Madden, T. L. 1997, Genome Res. 7:649-656,).

In one embodiment, the first or second binding partner (X or Y) is aprotein or peptide.

In one embodiment, the first and second fusion proteins comprise one ormore peptide linkers. The linkers may be incorporated at variouslocations in the fusion proteins. For example, a linker may beintroduced between a binding partner and the protein attached thereto.

In one embodiment, the linker is a peptide linker.

The term “peptide linker” as used herein refers to a peptide with aminoacid sequences. A range of suitable peptide linkers will be known to theperson of skill in the art.

In one embodiment, the peptide linker may be of synthetic origin, i.e.prepared by synthetic chemistry techniques.

In one embodiment, the binding partners of the bispecific proteincomplexes are joined to their respective proteins via peptide linkers.

In one embodiment the fusion proteins is a translational fusion, that isa fusion protein expressed in a host cells comprising a geneticconstruct from which the fusion protein is expressed.

In one embodiment the fusion protein is prepared by conjugating the A toX or B to Y optionally via a peptide linker.

In one embodiment, the peptide linker is 50 amino acids in length orless, for example 20 amino acids of less.

Generally it will be more efficient to express the fusion proteinrecombinantly and therefore a direct peptide bond or a peptide linkerthat can be expressed by a host cell may be advantageous.

In one aspect, there is provided a method of producing a bispecificprotein complex of the present disclosure, comprising the steps of:

-   -   (a) producing a first fusion protein (A-X), comprising a binding        domain specific to CD45 or CD79 a and/or CD79b (A), attached to        a first binding partner (X) of a binding pair;    -   (b) producing a second fusion protein (B-Y), comprising a        binding domain specific to CD45 or CD79a and/or CD79b (B),        attached to a second binding partner (Y) of a binding pair;        -   wherein at least the first fusion protein or the second            fusion protein comprises a binding domain specific to CD45            and the remaining fusion protein comprises a binding domain            specific to CD79a and/or CD79b, and    -   (c) mixing the first (A-X) and second fusion proteins (B-Y)        together prepared in step a) and b).

In particular, the heterodimerically-tethered bispecific protein complexis prepared by mixing A-X and B-Y in vitro. Thus in one embodiment themethod comprises an in vitro mixing step bringing A-X and B-Y intocontact.

Thus generally the fusion proteins A-X and B-Y are not co-expressed inthe same cell. This is advantageous because it allows, for example 100A-X fusion proteins and 100 A-Y fusion proteins to be expressedseparately and optionally purified, and through subsequent mixing of the200 fusion proteins in the various permutations can provide 10,000heterodimerically-tethered bispecific protein complexes.

In contrast prior art methods require co-expression of bispecifics andthus for 10,000 complexes, 10,000 transfections, expressions andpurifications are required.

The binding partners X and Y have affinity for each other and act asbiological equivalent of VELCRO or a bar and magnet and hold the complextogether. Advantageously, this means that the fusion proteins A-X andY-B can be readily assembled into a bispecific protein complex simply bymixing the fusion proteins together. Thus the bispecific protein complexof the present disclosure has a modular structure which allows for twodifferent proteins to be easily assembled in order to produce largepanels of permutations of bispecific protein complexes with differentcombinations of antigen specificities in, for example a grid-likefashion. This allows for the efficient and systematic screening of alarge number of bispecific protein complexes in order to detectadditive, synergistic or novel biological function.

Given X and Y are specific for each other this significantly reduces theability to form homodimers. X and Y are collectively referred to hereinas a binding pair or binding partners. In one embodiment X does not havehigh affinity for other Xs. In one embodiment Y does not have highaffinity for other Ys. Advantageously, when X and Y do not formhomodimers, this prevents the formation of undesired monospecificprotein complexes, increases yield of the desired bispecific proteincomplexes, and removes the need for onerous purification steps to removethe monospecific protein complexes.

This rapid assembly of bispecific protein complexes, the level of yieldand/or purity cannot be obtained efficiently by prior art methods, inparticular prior art methods generally require extensive purificationsteps.

Advantageously, the X and Y components allow a multiplex comprisingbispecific protein complexes made up of different permutations of fusionproteins to be assembled rapidly and easily.

In one embodiment the proteins A and B are antibodies or antibodyfragments. When the antibody or antibody fragments are held together asa complex via X and Y, this forms a bispecific antibody complex.

The mixing is generally effected in conditions where the X and Y caninteract. In one embodiment, the fusion proteins are incubated in cellculture media under cell culturing conditions, for example the fusionproteins are incubated for 90 minutes in a 37° C./5% CO₂ environment.

In one embodiment the fusions proteins of the present disclosure aremixed in an aqueous environment, for example one fusion protein may bebound to a solid surface such as a bead or a plate and the other fusionprotein can be introduced thereto in an aqueous solution/suspension. Thesolid phase allows excess components and reagents to be washed awayreadily. In one embodiment neither fusion is attached a solid phase andare simply admixed in a liquid/solution/medium.

Advantageously, the method of the present disclosure can be employed toprepare complexes formed between heterogenous pairs (i.e. between thefirst fusion protein [A-X] and second fusion protein [B-Y]) whereininteractions between homogenous pairs (i.e. between two first fusionproteins [A-X] or two second fusion proteins [B-Y]) are minimised. Thusthe present method allows large numbers of bispecific protein complexesto be prepared, with minimal or no contamination with homodimericcomplexes. This level of purity and yield is not possible using theprior art methods.

In one embodiment the complexes formed require no further purificationsteps.

In one embodiment the complexes formed require one purification step,for example column chromatography.

In one embodiment the method further comprises at least one purificationstep, for example after expression of a fusion protein according to thepresent disclosure.

A “functional assay,” as used herein, is an assay that can be used todetermine one or more desired properties or activities of the proteincomplexes, antibody complexes or the mixture of antibodies subject tothe assay conditions. Suitable functional assays may be binding assays,apoptosis assays, antibody-dependent cellular cytotoxicity (ADCC)assays, complement-dependent cytotoxicity (CDC) assays, inhibition ofcell growth or proliferation (cytostatic effect) assays, cell-killing(cytotoxic effect) assays, cell-signaling assays, cytokine productionassays, antibody production and isotype switching, and cellulardifferentiation assays, In one embodiment in vivo assays, such as animalmodels, including mouse tumor models, models of auto-immune disease,virus-infected or bacteria-infected rodent or primate models, and thelike, may be employed to test molecules of the present disclosure.

In the context of bispecific antibody complexes, the efficacy ofbispecific antibody complexes according to the present disclosure can becompared to individual antibodies or mixtures of antibodies (orfragments) in such models by methods generally known to one of ordinaryskill in the art.

The functional assays may be repeated a number of times as necessarywith or without different samples of a particular bispecific antibodycomplex to enhance the reliability of the results. Various statisticaltests known to the skilled person can be employed to identifystatistically significant results and thus identify bispecific antibodycomplexes with biological functions, and in particular to identifyoptimal variable region pairs for use in multispecific molecule of thepresent invention.

Compositions and Medical Uses

In one aspect there is provided a molecule according to the presentdisclosure or a component, such as a fusion protein, aheterodimerically-tethered bispecific protein complex, a compositioncomprising a molecule of the invention, including a fusion protein orsaid bispecific protein complex, a multiplex, array, library as definedherein.

In one embodiment the molecules of the present disclosure, for examplean antibody described herein, a multispecific molecule and/or abispecific protein complexes are suitable for therapeutic applicationsand may provide novel therapies for treating diseases. Thus in a furtheraspect, there is provided a molecule of the present disclosure, forexample a bispecific protein complex as described above, for use intherapy. The molecules of the present disclosure including themultispecific molecules and bispecific protein complexes describedherein are suitable for treating a range of diseases, such as cancer.

The molecules of the present disclosure, including the multispecificmolecules and bispecific protein complexes described herein are alsoparticularly suited for inhibiting B cell function in order to controlimmune and autoimmune reactions in various autoimmune diseases.

Thus, the present disclosure extends to a method of treating a diseasein a patient, comprising the administration of a therapeutically effectamount of a molecule of the present disclosure, for example amultispecific molecule or bispecific protein complex of the presentdisclosure. In one aspect, there is provided a pharmaceuticalcomposition comprising one or more molecules of the present disclosure,for example a multispecific molecule of the present disclosure.

Various different components can be included in the composition,including pharmaceutically acceptable carriers, excipients and/ordiluents. The composition may, optionally, comprise further moleculescapable of altering the characteristics of the population ofmultispecific molecules of the invention thereby, for example, reducing,stabilizing, delaying, modulating and/or activating the function of theantibodies. The composition may be in solid, or liquid form and may be,inter alia, be in the form of a powder, a tablet, a solution or anaerosol.

The present invention also provides a pharmaceutical or diagnosticcomposition comprising an antibody molecule or a multispecific moleculeof the present invention in combination with one or more of apharmaceutically acceptable excipient, diluent or carrier. Accordingly,provided is the use of a multispecific molecule of the invention for usein the treatment and for the manufacture of a medicament for thetreatment of a pathological condition or disorder.

Pathological Conditions

The pathological condition or disorder, may, for example be selectedfrom the group consisting of infections (viral, bacterial, fungal andparasitic), endotoxic shock associated with infection, arthritis such asrheumatoid arthritis, asthma such as severe asthma, chronic obstructivepulmonary disease (COPD), pelvic inflammatory disease, Alzheimer'sDisease, inflammatory bowel disease, Crohn's disease, ulcerativecolitis, Peyronie's Disease, coeliac disease, gallbladder disease,Pilonidal disease, peritonitis, psoriasis, vasculitis, surgicaladhesions, stroke, Type I Diabetes, lyme disease, meningoencephalitis,autoimmune uveitis, immune mediated inflammatory disorders of thecentral and peripheral nervous system such as multiple sclerosis, lupus(such as systemic lupus erythematosus) and Guillain-Barr syndrome,Atopic dermatitis, autoimmune hepatitis, fibrosing alveolitis, Grave'sdisease, IgA nephropathy, idiopathic thrombocytopenic purpura, Meniere'sdisease, pemphigus, primary biliary cirrhosis, sarcoidosis, scleroderma,Wegener's granulomatosis, other autoimmune disorders, pancreatitis,trauma (surgery), graft-versus-host disease, transplant rejection, heartdisease including ischaemic diseases such as myocardial infarction aswell as atherosclerosis, intravascular coagulation, bone resorption,osteoporosis, osteoarthritis, periodontitis, hypochlorhydia and cancer,including breast cancer, lung cancer, gastric cancer, ovarian cancer,hepatocellular cancer, colon cancer, pancreatic cancer, esophagealcancer, head & neck cancer, kidney, and cancer, in particular renal cellcarcinoma, prostate cancer, liver cancer, melanoma, sarcoma, myeloma,neuroblastoma, placental choriocarcinoma, cervical cancer, and thyroidcancer, and the metastatic forms thereof.

In one embodiment the disorder is cancer, for example Leukemia,including lyphocytic leukemia, such as acute lymphoblastic leukemia orchronic lymphocytic leukemia; or myelogenus leukemia, such as acturemyelogenous leukemia or chronic myelogenous leukemia.

In one embodiment autoimmune disease includes: —Acute disseminatedencephalomyelitis (adem), acute necrotizing hemorrhagicleukoencephalitis, Addison's disease, adrenal insufficiency,hypocortisolism, alopecia areata, amyloidosis, ankylosing spondylitis,spondyloarthritis, Strumpell-marie disease, anti-GBM/anti-TBM nephritis,antiphospholipid syndrome (aps), autoimmune angioedema, autoimmuneaplastic anemia, autoimmune dysautonomia, autoimmune hepatitis,autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune innerear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS),Canale-Smith syndrome, autoimmune myocarditis, autoimmune oophoritis,autoimmune pancreatitis (AIP), autoimmune polyglandular syndromes (typesI, II & III), autoimmune retinopathy (AR), autoimmune thrombocytopenicpurpura (ATP), autoimmune thyroid disease, autoimmune urticaria,axonal/neuronal neuropathies, balo disease, Behcet's disease, bullouspemphigoid, cardiomyopathy, Castleman disease, coeliac disease, chagasdisease, chronic inflammatory demyelinating polyneuropathy (CIDP),chronic recurrent multifocal ostomyelitis (CRMO), Churg-Strausssyndrome, cicatricial pemphigoid/benign mucosal pemphigoid (CP), Crohn'sdisease, inflammatory bowel disease, colitis, enteritis, ileitis, Coganssyndrome, cold agglutinin disease, congenital heart block, Coxsackiemyocarditis, crest disease, cryoglobulinemia, demyelinatingneuropathies, dermatitis herpetiformis, Duhring's disease,dermatomyositis, diabetes, type I, discoid lupus erythematosus (DLE),Dressler's syndrome, endometriosis, epidermolysis bullosa (EB) and ebacquisita (EBA), eosinophilic gastroenteritis, esophagitis, eosinophilicfasciitis, schulman's syndrome, erythema nodosum, experimental allergicencephalomyelitis, Evans syndrome, fibrosing alveolitis, giant cellarteritis (temporal arteritis), giant cell myocarditis,glomerulonephritis (non-proliferative: focal segmentalglomerulosclerosis and membranous glomerulonephritis. proliferative: IgAnephropathy), goodpasture's syndrome, granulomatosis with polyangiitis(GPA) (formerly called Wegener's granulomatosis), Graves' disease,Guillain-Barré syndrome, Miller Fisher syndrome, acute motor axonalneuropathy, acute motor sensory axonal neuropathy, acute panautonomicneuropathy, Bickerstaff's brainstem encephalitis, Hashimoto'sencephalitis, Hashimoto's thyroiditis, hemolytic anemia,Henoch-Schonlein purpura, herpes gestationis, hypogammaglobulinemia,idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura(ITP), IgA nephropathy (IGAN), berger's syndrome, synpharyngiticglomerulonephritis, IgA pemphigus, IgG4-related sclerosing disease,immune-regulated infertility, inclusion body myositis, insulin-dependentdiabetes mellitus, interstitial cystitis, Isaac's syndrome,neuromyotonia, juvenile arthritis, juvenile myositis, Kawasaki syndrome,Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus,lichen sclerosus, ligneous conjunctivitis, linear IgA dermatosis (LAD),pemphigoid, lupus (SLE), lyme disease, Meniere's disease, microscopicpolyangiitis (MPA), mixed connective tissue disease (MCTD), monoclonalgammaopathy, Mooren's ulcer, Mucha-Habermann disease, multiplesclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica(devic's), neuromyotonia, Isaac's syndrome (acquired, paraneoplastic,hereditary), neutropenia, ocular cicatricial pemphigoid, optic neuritis,oophoritis, opsoclonus-myoclonus syndrome, orchitis, palindromicrheumatism, pandas (pediatric autoimmune neuropsychiatric disordersassociated with streptococcus), paraneoplastic autoimmune multiorgansyndrome (PAMS), paraneoplastic cerebellar degeneration, paraneoplasticpemphigus (PNP), paroxysmal nocturnal hemoglobinuria (PNH), ParryRomberg syndrome, Parsonnage-Turner syndrome, pars planitis (peripheraluveitis), pempgigoid gestationis (PG), pemphigus vulgaris (PV),pemphigus folliaceus (PF), peripheral neuropathy, perivenousencephalomyelitis, pernicious anemia, Poems syndrome, polyarteritisnodosa (PAN), polymyalgia rheumatic, polymyositis, postmyocardialinfarction syndrome, postpericardiotomy syndrome, progesteronedermatitis primary biliary cirrhosis, Hanot syndrome, primary sclerosingcholangitis (PSC), sclerosong cholangitis, psoriasis, psoriaticarthritis, pyoderma gangrenosum, pure red cell aplasia, Rasmussen'sencephalitis, chronic focal encephalitis (CFE), Raynauds phenomenon,reactive arthritis, Reiter's syndrome, recoverin-associated retinopathy(RAR), reflex sympathetic dystrophy, Reiter's syndrome, relapsingpolychondritis, restless legs syndrome, retroperitoneal fibrosis,rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome,scleritis, scleroderma, systemic sclerosis, sjogren's syndrome, sperm &testicular autoimmunity, stiff person/man syndrome, subacute bacterialendocarditis (SBE), Susac's syndrome, sympathetic ophthalmia, Takayasu'sarteritis, temporal arteritis/giant cell arteritis, thromboangiitisobliterans, Buerger's disease, thrombocytopenic purpura (TTP),Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis,undifferentiated connective tissue disease (UCTD), uveitis, polymyalgiarheumatica, Takayasu's arteritis, temporal arteritis, Buerger's disease,cutaneous vasculitis, Kawasaki disease, polyarteritis nodosa, Behçet'ssyndrome, Churg-Strauss syndrome, cutaneous vasculitis, Henoch-Schonleinpurpura, microscopic polyangiitis, Wegener's granulomatosis, golfer'svasculitis, vesiculobullous dermatosis, Vitiligowegener's granulomatosis(now termed granulomatosis with polyangiitis (GPA).

In one embodiment the autoimmune disease is selected from the groupcomprising or consisting of: —ANCA vasculitis, IgA nephropathy(Berger's), pemphigus vulgaris/bullous pemphigoid, ITP, primary biliarycirrhosis, autoimmune thyroiditis (Grave's disease), hashimoto'sdisease, lupus nephritis, membranous glomerulonephritis (or membranousnephropathy), APS, myasthenia gravis, neuromyelitis optica, primarySjögren's, autoimmune neutropaenia, autoimmune pancreatitis,dermatosmyositis, autoimmune uveitis, autoimmune retinopathy, Behçet'sdisease, IPF, systemic sclerosis, liver fibrosis, autoimmune hepatitis,primary sclerosing cholangitis, vitiligo, goodpasture's syndrome,pulmonary alveolar proteinosis, chronic autoimmune urticarial,psoriasis, rheumatoid arthritis, psoriatic arthritis, axialspodyloarthritis, transplantation (including GvHD), asthma, COPD, giantcell arteritis, refractory autoimmune cytopaenias, Evans syndrome(autoimmune haemolytic anaemia), type I diabetes, sarcoidosis,polymyositis, ulcerative colitis, Crohn's disease, coeliac disease,Waldenstrom's macroglobulinaemia, focal segmental glomerulosclerosis,chronic Lyme disease (Lyme borreliosis), lichen planus, Stiff personsyndrome, dilated cardiomyopathy, autoimmune (lymphocytic) oophoritis,epidermolysis bullosa acquisita, autoimmune atrophic gastritis,pernicious anaemia, atopic dermatitis, atherosclerosis, multiplesclerosis, Rasmussen's encephalitis, Guillain-Barré syndrome andacquired neuromyotonia, stroke. In one embodiment the disorder iscancer, for example leukemia, for example lyphocytic leukemia, such asacute lymphoblastic leukemia or chronic lymphocytic leukemia; ormyelogenus leukemia, such as acture myelogenous leukemia or chronicmyelogenous leukemia; or lymphoma, such as diffuse large B cell lymphomaor Hodgkin's or non-Hodkin's lymphoma.

The present invention also provides a pharmaceutical or diagnosticcomposition comprising a molecule of the present disclosure, such as amultispecific molecule described herein in combination with one or moreof a pharmaceutically acceptable excipient, diluent or carrier.Accordingly, provided is the use of a molecule of the presentdisclosure, such as a multispecific molecule as described herein for usein treatment and in the manufacture of a medicament.

The composition will usually be supplied as part of a sterile,pharmaceutical composition that will normally include a pharmaceuticallyacceptable carrier. A pharmaceutical composition of the presentinvention may additionally comprise a pharmaceutically-acceptableadjuvant.

The present invention also provides a process for preparation of apharmaceutical or diagnostic composition comprising adding and mixingthe multispecific molecule of the present invention together with one ormore of a pharmaceutically acceptable excipient, diluent or carrier.

The term “pharmaceutically acceptable excipient” as used herein refersto a pharmaceutically acceptable formulation carrier, solution oradditive to enhance the desired characteristics of the compositions ofthe present disclosure. Excipients are well known in the art and includebuffers (e.g., citrate buffer, phosphate buffer, acetate buffer andbicarbonate buffer), amino acids, urea, alcohols, ascorbic acid,phospholipids, proteins (e.g., serum albumin), EDTA, sodium chloride,liposomes, mannitol, sorbitol, and glycerol. Solutions or suspensionscan be encapsulated in liposomes or biodegradable microspheres. Theformulation will generally be provided in a substantially sterile formemploying sterile manufacture processes.

This may include production and sterilization by filtration of thebuffered solvent solution used for the formulation, aseptic suspensionof the antibody in the sterile buffered solvent solution, and dispensingof the formulation into sterile receptacles by methods familiar to thoseof ordinary skill in the art.

The pharmaceutically acceptable carrier should not itself induce theproduction of antibodies harmful to the individual receiving thecomposition and should not be toxic. Suitable carriers may be large,slowly metabolised macromolecules such as proteins, polypeptides,liposomes, polysaccharides, polylactic acids, polyglycolic acids,polymeric amino acids, amino acid copolymers and inactive virusparticles.

Pharmaceutically acceptable salts can be used, for example mineral acidsalts, such as hydrochlorides, hydrobromides, phosphates and sulphates,or salts of organic acids, such as acetates, propionates, malonates andbenzoates.

Pharmaceutically acceptable carriers in therapeutic compositions mayadditionally contain liquids such as water, saline, glycerol andethanol. Such carriers enable the pharmaceutical compositions to beformulated as tablets, pills, dragées, capsules, liquids, gels, syrups,slurries and suspensions, for ingestion by the patient.

The molecules of the disclosure such as a multispecific moleculedescribed herein can be delivered dispersed in a solvent, e.g., in theform of a solution or a suspension. It can be suspended in anappropriate physiological solution, e.g., physiological saline, apharmacologically acceptable solvent or a buffered solution. Bufferedsolutions known in the art may contain 0.05 mg to 0.15 mg disodiumedetate, 8.0 mg to 9.0 mg NaCl, 0.15 mg to 0.25 mg polysorbate, 0.25 mgto 0.30 mg anhydrous citric acid, and 0.45 mg to 0.55 mg sodium citrateper 1 ml of water so as to achieve a pH of about 4.0 to 5.0. Asmentioned supra a suspension can made, for example, from lyophilisedantibody.

A thorough discussion of pharmaceutically acceptable carriers isavailable in Remington's Pharmaceutical Sciences (Mack PublishingCompany, N.J. 1991).

The term “therapeutically effective amount” as used herein refers to anamount of a therapeutic agent needed to treat, ameliorate or prevent atargeted disease or condition, or to exhibit a detectable therapeutic orpreventative effect. For any antibody, the therapeutically effectiveamount can be estimated initially either in cell culture assays or inanimal models, usually in rodents, rabbits, dogs, pigs or primates. Theanimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

The precise therapeutically effective amount for a human subject willdepend upon the severity of the disease state, the general health of thesubject, the age, weight and gender of the subject, diet, time andfrequency of administration, drug combination(s), reaction sensitivitiesand tolerance/response to therapy. This amount can be determined byroutine experimentation and is within the judgement of the clinician.Generally, a therapeutically effective amount will be from 0.01 mg/kg to50 mg/kg, for example 0.1 mg/kg to 20 mg/kg. Alternatively, the dose maybe 1 to 500 mg per day such as 10 to 100, 200, 300 or 400 mg per day.Pharmaceutical compositions may be conveniently presented in unit doseforms containing a predetermined amount of an active agent of theinvention.

Compositions may be administered individually to a patient or may beadministered in combination (e.g. simultaneously, sequentially orseparately) with other agents, drugs or hormones.

The dose at which the multispecific molecule of the present disclosureis administered depends on the nature of the condition to be treated,the extent of the inflammation present and on whether the antibodymolecule is being used prophylactically or to treat an existingcondition. The frequency of dose will depend on the half-life of themultispecific molecule and the duration of its effect. If themultispecific molecule has a short half-life (e.g. 2 to 10 hours) it maybe necessary to give one or more doses per day. Alternatively, if themultispecific molecule has a long half-life (e.g. 2 to 15 days) it mayonly be necessary to give a dosage once per day, once per week or evenonce every 1 or 2 months.

In the present disclosure, the pH of the final formulation is notsimilar to the value of the isoelectric point of the multispecificmolecule, for if the pH of the formulation is 7 then a pI of from 8-9 orabove may be appropriate. Whilst not wishing to be bound by theory it isthought that this may ultimately provide a final formulation withimproved stability, for example the antibody or fragment remains insolution.

The pharmaceutical compositions of this invention may be administered byany number of routes including, but not limited to, oral, intravenous,intramuscular, intra-arterial, intramedullary, intrathecal,intraventricular, transdermal, transcutaneous (for example, seeWO98/20734), subcutaneous, intraperitoneal, intranasal, enteral,topical, sublingual, intravaginal or rectal routes. Hyposprays may alsobe used to administer the pharmaceutical compositions of the invention.

Direct delivery of the compositions will generally be accomplished byinjection, subcutaneously, intraperitoneally, intravenously orintramuscularly, or delivered to the interstitial space of a tissue. Thecompositions can also be administered into a specific tissue ofinterest. Dosage treatment may be a single dose schedule or a multipledose schedule.

Where the product is for injection or infusion, it may take the form ofa suspension, solution or emulsion in an oily or aqueous vehicle and itmay contain formulatory agents, such as suspending, preservative,stabilising and/or dispersing agents. Alternatively, the multispecificmolecule may be in dry form, for reconstitution before use with anappropriate sterile liquid. If the composition is to be administered bya route using the gastrointestinal tract, the composition will need tocontain agents which protect the antibody from degradation but whichrelease the bispecific protein complex once it has been absorbed fromthe gastrointestinal tract. A nebulisable formulation according to thepresent disclosure may be provided, for example, as single dose units(e.g., sealed plastic containers or vials) packed in foil envelopes.Each vial contains a unit dose in a volume, e.g., 2 ml, ofsolvent/solution buffer.

The term “variant” as used herein refers to peptide or protein thatcontains at least one amino acid sequence or nucleotide sequencealteration as compared to the amino acid or nucleotide sequence of thecorresponding wild-type peptide or protein. A variant may comprise atleast 80%, or 85%, or 90%, or 95%, or 98% or 99% sequence identity tothe corresponding wild-type peptide or protein. However, it is possiblefor a variant to comprise less than 80% sequence identity, provided thatthe variant exhibits substantially similar function to its correspondingwild-type peptide or protein.

In one embodiment the construct of the present disclosure is at leasttrispecific. In this situation the further specificity may be directedto any antigen of interest, for example antigens to extend half-lifesuch as albumin or Fc neonatal receptor (FcRn); antigens for effectorfunction such as activating or inhibiting Fc receptors or costimulatorymolecules; tissue or cell targeting antigens; or antigens to aidblood/brain barrier (BBB) transfer such as transferrin receptor or LRP1.

The disclosure also extends to compositions, such as pharmaceuticalcompositions comprising said novel formats with the particular antigenspecificity.

In a further aspect the disclosure includes use of the formats and thecompositions in treatment. The present invention also provides a processfor preparation of a pharmaceutical or diagnostic composition comprisingadding and mixing the antibody molecule of the present inventiontogether with one or more of a pharmaceutically acceptable excipient,diluent or carrier.

The antibody molecule may be the sole active ingredient in thepharmaceutical or diagnostic composition or may be accompanied by otheractive ingredients including other antibody ingredients or non-antibodyingredients such as steroids or other drug molecules.

The pharmaceutical compositions suitably comprise a therapeuticallyeffective amount of the antibody of the invention. The term“therapeutically effective amount” as used herein refers to an amount ofa therapeutic agent needed to treat, ameliorate or prevent a targeteddisease or condition, or to exhibit a detectable therapeutic orpreventative effect. For any antibody, the therapeutically effectiveamount can be estimated initially either in cell culture assays or inanimal models, usually in rodents, rabbits, dogs, pigs or primates. Theanimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

The precise therapeutically effective amount for a human subject willdepend upon the severity of the disease state, the general health of thesubject, the age, weight and gender of the subject, diet, time andfrequency of administration, drug combination(s), reaction sensitivitiesand tolerance/response to therapy. This amount can be determined byroutine experimentation and is within the judgement of the clinician.Generally, a therapeutically effective amount will be from 0.01 mg/kg to500 mg/kg, for example 0.1 mg/kg to 200 mg/kg, such as 100 mg/Kg.Pharmaceutical compositions may be conveniently presented in unit doseforms containing a predetermined amount of an active agent of theinvention per dose.

Compositions may be administered individually to a patient or may beadministered in combination (e.g. simultaneously, sequentially orseparately) with other agents, drugs or hormones.

Agents as employed herein refers to an entity which when administeredhas a physiological affect.

Drug as employed herein refers to a chemical entity which at atherapeutic dose has an appropriate physiological affect.

In one embodiment the antibodies or fragments according to the presentdisclosure are employed with an immunosuppressant therapy, such as asteroid, in particular prednisone.

In one embodiment the antibodies or fragments according to the presentdisclosure are employed with Rituximab or other B cell therapies.

In one embodiment the antibodies or fragments according to the presentdisclosure are employed with any B cell or T cell modulating agent orimmunomodulator. Examples include methotrexate, microphenyolate andazathioprine.

The dose at which the antibody molecule of the present invention isadministered depends on the nature of the condition to be treated, theextent of the inflammation present and on whether the antibody moleculeis being used prophylactically or to treat an existing condition.

The frequency of dose will depend on the half-life of the antibodymolecule and the duration of its effect. If the antibody molecule has ashort half-life (e.g. 2 to 10 hours) it may be necessary to give one ormore doses per day. Alternatively, if the antibody molecule has a longhalf life (e.g. 2 to 15 days) and/or long lasting pharmacodynamics (PD)profile it may only be necessary to give a dosage once per day, once perweek or even once every 1 or 2 months.

In one embodiment the dose is delivered bi-weekly, i.e. twice a month.

In one embodiment doses are spaced to allow anti-drug (in this caseanti-antibody) responses to waine before administration of further dose.

Half life as employed herein is intended to refer to the duration of themolecule in circulation, for example in serum/plasma.

Pharmacodynamics as employed herein refers to the profile and inparticular duration of the biological action of the molecule accordingthe present disclosure.

The pharmaceutically acceptable carrier should not itself induce theproduction of antibodies harmful to the individual receiving thecomposition and should not be toxic. Suitable carriers may be large,slowly metabolised macromolecules such as proteins, polypeptides,liposomes, polysaccharides, polylactic acids, polyglycolic acids,polymeric amino acids, amino acid copolymers and inactive virusparticles.

Pharmaceutically acceptable salts can be used, for example mineral acidsalts, such as hydrochlorides, hydrobromides, phosphates and sulphates,or salts of organic acids, such as acetates, propionates, malonates andbenzoates.

Pharmaceutically acceptable carriers in therapeutic compositions mayadditionally contain liquids such as water, saline, glycerol andethanol. Additionally, auxiliary substances, such as wetting oremulsifying agents or pH buffering substances, may be present in suchcompositions. Such carriers enable the pharmaceutical compositions to beformulated as tablets, pills, dragees, capsules, liquids, gels, syrups,slurries and suspensions, for ingestion by the patient.

Suitable forms for administration include forms suitable for parenteraladministration, e.g. by injection or infusion, for example by bolusinjection or continuous infusion. Where the product is for injection orinfusion, it may take the form of a suspension, solution or emulsion inan oily or aqueous vehicle and it may contain formulatory agents, suchas suspending, preservative, stabilising and/or dispersing agents.Alternatively, the antibody molecule may be in dry form, forreconstitution before use with an appropriate sterile liquid.

Once formulated, the compositions of the invention can be administereddirectly to the subject. The subjects to be treated can be animals.However, in one or more embodiments the compositions are adapted foradministration to human subjects.

Suitably in formulations according to the present disclosure, the pH ofthe final formulation is not similar to the value of the isoelectricpoint of the antibody or fragment, for example if the pI of the proteinis in the range 8-9 or above then a formulation pH of 7 may beappropriate. Whilst not wishing to be bound by theory it is thought thatthis may ultimately provide a final formulation with improved stability,for example the antibody or fragment remains in solution. In one examplethe pharmaceutical formulation at a pH in the range of 4.0 to 7.0comprises: 1 to 200 mg/mL of an antibody molecule according to thepresent disclosure, 1 to 100 mM of a buffer, 0.001 to 1% of asurfactant, a) 10 to 500 mM of a stabiliser, b) 10 to 500 mM of astabiliser and 5 to 500 mM of a tonicity agent, or c) 5 to 500 mM of atonicity agent.

The pharmaceutical compositions of this invention may be administered byany number of routes including, but not limited to, oral, intravenous,intramuscular, intra-arterial, intramedullary, intrathecal,intraventricular, transdermal, transcutaneous (for example, seeWO98/20734), subcutaneous, intraperitoneal, intranasal, enteral,topical, sublingual, intravaginal or rectal routes. Hyposprays may alsobe used to administer the pharmaceutical compositions of the invention.Typically, the therapeutic compositions may be prepared as injectables,either as liquid solutions or suspensions. Solid forms suitable forsolution in, or suspension in, liquid vehicles prior to injection mayalso be prepared.

Direct delivery of the compositions will generally be accomplished byinjection, subcutaneously, intraperitoneally, intravenously orintramuscularly, or delivered to the interstitial space of a tissue. Thecompositions can also be administered into a lesion. Dosage treatmentmay be a single dose schedule or a multiple dose schedule.

It will be appreciated that the active ingredient in the compositionwill be an antibody molecule. As such, it will be susceptible todegradation in the gastrointestinal tract. Thus, if the composition isto be administered by a route using the gastrointestinal tract, thecomposition will need to contain agents which protect the antibody fromdegradation but which release the antibody once it has been absorbedfrom the gastrointestinal tract.

A thorough discussion of pharmaceutically acceptable carriers isavailable in Remington's Pharmaceutical Sciences (Mack PublishingCompany, N.J. 1991).

In one embodiment the formulation is provided as a formulation fortopical administrations including inhalation.

Suitable inhalable preparations include inhalable powders, meteringaerosols containing propellant gases or inhalable solutions free frompropellant gases. Inhalable powders according to the disclosurecontaining the active substance may consist solely of the abovementionedactive substances or of a mixture of the abovementioned activesubstances with physiologically acceptable excipient.

These inhalable powders may include monosaccharides (e.g. glucose orarabinose), disaccharides (e.g. lactose, saccharose, maltose), oligo-and polysaccharides (e.g. dextranes), polyalcohols (e.g. sorbitol,mannitol, xylitol), salts (e.g. sodium chloride, calcium carbonate) ormixtures of these with one another. Mono- or disaccharides are suitablyused, the use of lactose or glucose, particularly but not exclusively inthe form of their hydrates.

Particles for deposition in the lung require a particle size less than10 microns, such as 1-9 microns for example from 1 to 5 μm. The particlesize of the active ingredient (such as the antibody or fragment) is ofprimary importance.

The propellent gases which can be used to prepare the inhalable aerosolsare known in the art. Suitable propellent gases are selected from amonghydrocarbons such as n-propane, n-butane or isobutane andhalohydrocarbons such as chlorinated and/or fluorinated derivatives ofmethane, ethane, propane, butane, cyclopropane or cyclobutane. Theabovementioned propellent gases may be used on their own or in mixturesthereof.

Particularly suitable propellent gases are halogenated alkanederivatives selected from among TG 11, TG 12, TG 134a and TG227. Of theabovementioned halogenated hydrocarbons, TG134a(1,1,1,2-tetrafluoroethane) and TG227 (1,1,1,2,3,3,3-heptafluoropropane)and mixtures thereof are particularly suitable.

The propellent-gas-containing inhalable aerosols may also contain otheringredients such as cosolvents, stabilisers, surface-active agents(surfactants), antioxidants, lubricants and means for adjusting the pH.All these ingredients are known in the art.

The propellant-gas-containing inhalable aerosols according to theinvention may contain up to 5% by weight of active substance. Aerosolsaccording to the invention contain, for example, 0.002 to 5% by weight,0.01 to 3% by weight, 0.015 to 2% by weight, 0.1 to 2% by weight, 0.5 to2% by weight or 0.5 to 1% by weight of active ingredient.

Alternatively topical administrations to the lung may also be byadministration of a liquid solution or suspension formulation, forexample employing a device such as a nebulizer, for example, a nebulizerconnected to a compressor (e.g., the Pari LC-Jet Plus® nebulizerconnected to a Pari Master® compressor manufactured by Pari RespiratoryEquipment, Inc., Richmond, Va.).

The antibody or multispecific molecule of the invention can be delivereddispersed in a solvent, e.g., in the form of a solution or a suspension.It can be suspended in an appropriate physiological solution, e.g.,saline or other pharmacologically acceptable solvent or a bufferedsolution. Buffered solutions known in the art may contain 0.05 mg to0.15 mg disodium edetate, 8.0 mg to 9.0 mg NaCl, 0.15 mg to 0.25 mgpolysorbate, 0.25 mg to 0.30 mg anhydrous citric acid, and 0.45 mg to0.55 mg sodium citrate per 1 ml of water so as to achieve a pH of about4.0 to 5.0. A suspension can employ, for example, lyophilised antibody.

The therapeutic suspensions or solution formulations can also containone or more excipients. Excipients are well known in the art and includebuffers (e.g., citrate buffer, phosphate buffer, acetate buffer andbicarbonate buffer), amino acids, urea, alcohols, ascorbic acid,phospholipids, proteins (e.g., serum albumin), EDTA, sodium chloride,liposomes, mannitol, sorbitol, and glycerol. Solutions or suspensionscan be encapsulated in liposomes or biodegradable microspheres. Theformulation will generally be provided in a substantially sterile formemploying sterile manufacture processes.

This may include production and sterilization by filtration of thebuffered solvent/solution used for the formulation, aseptic suspensionof the antibody in the sterile buffered solvent solution, and dispensingof the formulation into sterile receptacles by methods familiar to thoseof ordinary skill in the art.

Nebulizable formulation according to the present disclosure may beprovided, for example, as single dose units (e.g., sealed plasticcontainers or vials) packed in foil envelopes. Each vial contains a unitdose in a volume, e.g., 2 mL, of solvent/solution buffer.

The antibodies disclosed herein may be suitable for delivery vianebulisation.

It is also envisaged that the antibody of the present invention may beadministered by use of gene therapy. In order to achieve this, DNAsequences encoding the heavy and light chains of the antibody moleculeunder the control of appropriate DNA components are introduced into apatient such that the antibody chains are expressed from the DNAsequences and assembled in situ.

In one embodiment, the molecule of the present disclosure, such as abispecific protein complex described herein may be used to functionallyalter the activity of the antigen or antigens of interest. For example,the bispecific protein complex may neutralize, antagonize or agonise theactivity of said antigen or antigens, directly or indirectly.

The present disclosure also extends to a kit, comprising a molecule ofthe present disclosure or a component thereof. In one embodiment the kitcomprises:

-   a) one or more fusion proteins (A-X) comprising a first antibody or    antibody fragment (A) specific to CD45 or CD79a and/or CD79b    attached to a first binding partner of a binding pair (X); and-   b) one or more fusion proteins (B-Y) comprising a second antibody or    antibody fragment

(B) specific to CD45 or CD79a and/or CD79b attached to a second bindingpartner of the binding pair (Y), wherein the latter is specific for thefirst binding partner; for example wherein the first binding partner (X)is a peptide or polypeptide and the second binding (Y) partner is anantibody or antibody fragment specific thereto;

wherein Y the second binding partner is specific to the first bindingpartner X and the second binding partner is, for example an antibody orantibody fragment specific thereto; and the specific interaction (suchas a binding interaction) of the two binding partners forms aheterodimer-tether which physically brings the two fusion proteins froma) and b) together to form a bispecific protein complex; andwherein at least one of A or B is specific to CD45 and the other isspecific to CD79a and/or CD79b, andthe fusion protein(s) is/are in a complexed or a non-complexed form.

Advantageously, the kit may comprise bispecific protein complexes of thepresent disclosure, or may comprise fusion proteins which are in acomplexed or non-complexed form. In the former case, the bispecificprotein complexes are ready for use “out of the box” which providesconvenience and ease of use, whereas in the latter case, the bispecificprotein complexes can be assembled according to the user's requirementsby using combining different fusion proteins.

In another embodiment, the kit further comprises instructions for use.

In yet another embodiment, the kit further comprises one or morereagents for performing one or more functional assays.

In one embodiment, molecules of the present disclosure including fusionproteins, bispecific proteins complexes or compositions comprising sameare provided for use as a laboratory reagent.

Further Aspects

In a further aspect, there is provided a nucleotide sequence, forexample a DNA sequence encoding a construct as described hereinincluding a multispecific molecule or a fusion protein as defined above.

In one embodiment, there is provided a nucleotide sequence, for examplea DNA sequence encoding a construct as described herein including amultispecific molecule or a bispecific protein complex or an antibodyaccording to the present disclosure.

The disclosure herein also extends to a vector comprising a nucleotidesequence as defined above.

The term “vector” as used herein refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. An example of a vector is a “plasmid,” which is a circulardouble stranded DNA loop into which additional DNA segments may beligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell, where they are subsequently replicated along with the hostgenome. In the present specification, the terms “plasmid” and “vector”may be used interchangeably as a plasmid is the most commonly used formof vector.

General methods by which the vectors may be constructed, transfectionmethods and culture methods are well known to those skilled in the art.In this respect, reference is made to “Current Protocols in MolecularBiology”, 1999, F. M. Ausubel (ed), Wiley Interscience, New York and theManiatis Manual produced by Cold Spring Harbor Publishing.

The term “selectable marker” as used herein refers to a protein whoseexpression allows one to identify cells that have been transformed ortransfected with a vector containing the marker gene. A wide range ofselection markers are known in the art. For example, typically theselectable marker gene confers resistance to drugs, such as G418,hygromycin or methotrexate, on a host cell into which the vector hasbeen introduced. The selectable marker can also be a visuallyidentifiable marker such as a fluorescent marker for example. Examplesof fluorescent markers include rhodamine, FITC, TRITC, Alexa Fluors andvarious conjugates thereof.

Also provided is a host cell comprising one or more cloning orexpression vectors comprising one or more DNA sequences encoding anantibody of the present disclosure. Any suitable host cell/vector systemmay be used for expression of the DNA sequences encoding the antibodymolecule of the present disclosure. Bacterial, for example E. coli, andother microbial systems may be used or eukaryotic, for examplemammalian, host cell expression systems may also be used. Suitablemammalian host cells include CHO, myeloma or hybridoma cells.

The present disclosure also provides a process for the production of amolecule according to the present disclosure or a component thereofcomprising culturing a host cell containing a vector of the presentdisclosure under conditions suitable for leading to expression ofprotein from DNA encoding the molecule of the present disclosure, andisolating the molecule.

The molecules of the present disclosure including the bispecific proteincomplexes described herein may be used in diagnosis/detection kits. Thekits may, for example comprise bispecific antibody complexes that arespecific for two antigens, both of which are present on the same celltype, and wherein a positive diagnosis can only be made if both antigensare successfully detected. By using a molecule of the present disclosuresuch as a bispecific antibody complexes described herein rather than twoseparate antibodies or antibody fragments in a non-complexed form, thespecificity of the detection can be greatly enhanced.

In one embodiment, the molecules of the present disclosure such as thebispecific antibody complexes are fixed on a solid surface. The solidsurface may for example be a chip, or an ELISA plate.

Further provided is the use of a molecule according to the presentdisclosure, for example a bispecific protein complex described hereinfor detecting in a sample the presence of a first and a second peptide,whereby the said molecules are used as detection agents.

The molecules of the present disclosure such as the bispecific antibodycomplexes described herein may for example be conjugated to afluorescent marker which facilitates the detection of boundantibody-antigen complexes. Such bispecific antibody complexes can beused for immunofluorescence microscopy. Alternatively, the bispecificantibody complexes may also be used for western blotting or ELISA.

In one embodiment, there is provided a process for purifying a moleculeaccording to the present disclosure or a component thereof

In one embodiment, there is provided a process for purifying a moleculeaccording the present disclosure or a component thereof comprising thesteps: performing anion exchange chromatography in non-binding mode suchthat the impurities are retained on the column and the antibody ismaintained in the unbound fraction. The step may, for example beperformed at a pH about 6-8.

The process may further comprise an initial capture step employingcation exchange chromatography, performed for example at a pH of about 4to 5.

The process may further comprise of additional chromatography step(s) toensure product and process related impurities are appropriately resolvedfrom the product stream.

The purification process may also comprise of one or moreultra-filtration steps, such as a concentration and diafiltration step.

“Purified form” as used supra is intended to refer to at least 90%purity, such as 91, 92, 93, 94, 95, 96, 97, 98, 99% w/w or more pure.

In the context of this specification “comprising” is to be interpretedas “including”.

Aspects of the disclosure comprising certain elements are also intendedto extend to alternative embodiments “consisting” or “consistingessentially” of the relevant elements.

Positively recited embodiments may be employed herein as a basis for adisclaimer.

All references referred to herein are specifically incorporated byreference.

The sub-headings herein are employed to assist in structuring thespecification and are not intended to be used to construct the meaningof technical terms herein.

Sequences of the disclosure are provided herein below.

GCN4(7P14P) sequences SEQ ID NO: 1ASGGGRMKQLEPKVEELLPKNYHLENEVARLKKLVGERHHHHHHwherein the amino acids in bold are optional SEQ ID NO: 2GCTAGCGGAGGCGGAAGAATGAAACAACTTGAACCCAAGGTTGAAGAATTGCTTCCGAAAAATTATCACTTGGAAAATGAGGTTGCCAGATTAAAGAAATTAGTTGGCGAACGCCATCACCATCACCATCAC 52SR4 ds scFv- sequence SEQ ID NO: 3DAVVTQESALTSSPGETVTLTCRSSTGAVTTSNYASWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCVLWYSDHWVFGCGTKLTVLGGGGGSGGGGSGGGGSGGGGSDVQLQQSGPGLVAPSQSLSITCTVSGFLLTDYGVNWVRQSPGKCLEWLGVIWGDGITDYNSALKSRLSVTKDNSKSQVFLKMNSLQSGDSARYYCVTGLFDYWGQGTTLTVSSAAAHHHH HHEQKLISEEDL-SEQ ID NO: 4 GATGCGGTGGTGACCCAGGAAAGCGCGCTGACCAGCAGCCCGGGCGAAACCGTGACCCTGACCTGCCGCAGCAGCACCGGCGCGGTGACCACCAGCAACTATGCGAGCTGGGTGCAGGAAAAACCGGATCATCTGTTTACCGGCCTGATTGGCGGCACCAACAACCGCGCGCCGGGCGTGCCGGCGCGCTTTAGCGGCAGCCTGATTGGCGATAAAGCGGCGCTGACCATTACCGGCGCGCAGACCGAAGATGAAGCGATTTATTTTTGCGTGCTGTGGTATAGCGACCATTGGGTGTTTGGCTGCGGCACCAAACTGACCGTGCTGGGTGGAGGCGGTGGCTCAGGCGGAGGTGGCTCAGGCGGTGGCGGGTCTGGCGGCGGCGGCAGCGATGTGCAGCTGCAGCAGAGCGGCCCGGGCCTGGTGGCGCCGAGCCAGAGCCTGAGCATTACCTGCACCGTGAGCGGCTTTCTCCTGACCGATTATGGCGTGAACTGGGTGCGCCAGAGCCCGGGCAAATGCCTGGAATGGCTGGGCGTGATTTGGGGCGATGGCATTACCGATTATAACAGCGCGCTGAAAAGCCGCCTGAGCGTGACCAAAGATAACAGCAAAAGCCAGGTGTTTCTGAAAATGAACAGCCTGCAGAGCGGCGATAGCGCGCGCTATTATTGCGTGACCGGCCTGTTTGATTATTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGCGGCCGCCCATCACCATCACCATCACGAACAGAAACTGATTAGCGAAGAAGATCTGTAATAG

CD79b Antibodies

Ab 4447 Rabbit Ab 4447 VL region SEQ ID NO: 71AQVLTQTPSP VSAPVGGTVT INCQASQSVV SGNYLAWLQQKPGQPPKQLI HSASTLASGV SSRFSGSGS G TQFTLTISGV QCEDAATYYC LGEFSCSSHDCNAFGGGTEV VVK Rabbit Ab 4447 VL region SEQ ID NO: 72gcccaagtgc tgacccagac tccgtcccct gtgtctgcacctgtgggagg cacagtcacc atcaattgcc aggccagtcagagtgttgtt agtggcaatt acctagcctg gcttcagcagaaaccagggc agcctcccaa gcaactgatc cattctgcatccactctggc atctggggtc tcatcgcggt tcagcggcagtggatctggg acacaattca ctctcaccat cagcggcgtgcagtgtgaag atgctgccac ttactactgt ctaggcgaatttagttgtag tagtcatgat tgtaatgctt tcggcggagg gaccgaggtg gtggtcaaaRabbit Ab 4447 VH region SEQ ID NO: 73QSLEESGGRL VTPGTPLTLT CTVSGFSLSN YAVSWVRQAPGEGLEWIGII YIETGTTWYA NWAKGRFTIS KTSTTVDLTITSPSTEDTAT YFCAREPYEP YDDSNIYYGM DPWGPGTLVT VSS Rabbit Ab 4447 VH regionSEQ ID NO: 74 cagtcgctgg aggagtccgg gggtcgcctg gtcacgcctgggacacccct gacactcacc tgcaccgtct ctggattctccctcagtaac tatgcagtaa gctgggtccg ccaggctccaggggagggac tggaatggat cgggatcatt tatattgaaactggtaccac atggtacgcg aactgggcga aaggccgattcaccatctcc aaaacctcga ccacggtgga tctgacaatcaccagtccgt caaccgagga cacggccacc tatttctgtgccagagaacc ttatgaacct tatgatgata gtaatatttactacggcatg gacccctggg gcccaggcac cctcgtcacc gtctcgagt CDRL1SEQ ID NO: 75 QASQSVVSGNYLA CDRL2 SEQ ID NO: 76 SASTLAS CDRL3SEQ ID NO: 77 LGEFSCSSHDCNA CDRH1 SEQ ID NO: 78 GFSLSNYAVS CDRH2SEQ ID NO: 79 IIYIETGTTWYANWAKG CDRH3 SEQ ID NO: 80 EPYEPYDDSNIYYGMDP

The disclosure also extends to a derivative of SEQ ID NO: 77 wherein oneor both cysteine are replaced with another amino acid for exampleserine, in particular where the first cys is replaced by serine and thesecond cys remains unchanged, or the first cysteine remains unchangedand the second cysteine is replaced by serine, or where both cysteinesare replaced by serine.

Ab 4450 Rabbit Ab 4450 VL region SEQ ID NO: 81AIDMTQTPSP VSAAVGGTVT INCQSSQSIY NNNDLAWYQQKPGQPPKLLI YEASKLASGV PSRFKGSGSG TQFTLTISGVQCDDAATYYC QGGGSGGDGI AFGGGTKVVV E Rabbit Ab 4450 VL regionSEQ ID NO: 82 gccattgata tgacccagac tccatccccc gtgtctgcagctgtgggagg cacagtcacc atcaattgcc agtccagtcagagtatttat aataataatg acttagcctg gtatcagcagaaaccagggc agcctcccaa gctcctgatc tacgaagcatccaaactggc atctggggtc ccatcgcggt tcaaaggcagtggatctggg acacagttca ctctcaccat cagtggcgtgcagtgtgatg atgctgccac ttactactgt cagggcggtggtagtggtgg tgatggcatt gctttcggcg gagggaccaa ggtggtcgtc gaaRabbit Ab 4450 VH region SEQ ID NO: 83QSVEESGGRL VTPGAPLTLT CTVSGFSLNN YVMVWVRQAPGKGLEWIGII YVSGNAYYAS WAKGRFTISR TSTTVDLKVTSLTTEDTATY FCARDAGHSD VDVLDIWGPG TLVTVSS Rabbit Ab 4450 VH regionSEQ ID NO: 84 cagtcggtgg aggagtccgg gggtcgcctg gtcacgcctggggcacccct gacactcacc tgcacagtct ctggattctccctcaataac tatgtaatgg tctgggtccg ccaggctccagggaaggggc tggaatggat cggaatcatt tatgttagtggtaatgcata ctacgcgagc tgggcaaaag gccgattcaccatctccaga acctcgacca cggtggatct gaaagtgaccagtctgacaa ccgaggacac ggccacctat ttctgtgccagagatgctgg tcatagtgat gtcgatgttt tggatatttggggcccgggc accctcgtca ccgtctcgag t CDRL1 SEQ ID NO: 85 QSSQSIYNNNDLACDRL2 SEQ ID NO: 86 EASKLAS CDRL3 SEQ ID NO: 87 QGGGSGGDGIA CDRH1SEQ ID NO: 88 GFSLNNYVMV CDRH2 SEQ ID NO: 89 IIYVSGNAYYASWAKG CDRH3SEQ ID NO: 90 DAGHSDVDVLDI

The disclosure also extends to a derivative of SEQ ID NO: 87 wherein atleast one of the amino acids in the motif DG is replaced by anotheramino acid, for example the motif is mutated to EG, DA or DS.

CD45 Antibodies

Ab 4122 Rabbit Ab 4122 VL region SEQ ID NO: 91DIVMTQTPAS VSEPVGGTVT IMCQASQSIS NWLAWYQQKPGQPPKLLIYQ ASKLASGVPS RFKGSGSGTE YTLTISDLECADAATYYCQS YYDSGSNVFF AFGGGTKVVV E Rabbit Ab 4122 VL regionSEQ ID NO: 92 gacattgtga tgacccagac tccagcctcc gtgtctgaacctgtgggagg cacagtcacc atcatgtgcc aggccagtcagagcattagc aattggttag cctggtatca acagaaaccagggcagcctc ccaagctcct gatctaccag gcatccaaactggcatctgg ggtcccatcg cggttcaaag gcagtggatctgggacagag tacactctca ccatcagcga cctggagtgtgccgatgctg ccacttacta ctgtcaaagc tattatgatagtggtagtaa tgtttttttt gctttcggcg gagggaccaa ggtggtggtc gaaRabbit Ab 4122 VH region SEQ ID NO: 93LSLEESGGDL VKPGASLTLT CTASGFSFSA GYWICWVRQAPGKGLEWIAC TYAGRSGSTY YANWVNGRFT IPKTSSTTVTLQMTSLSGAD TASYFCARGN AGVAVGALWG PGTLVTVSS Rabbit Ab 4122 VH regionSEQ ID NO: 94 ctgtcgttgg aggagtccgg gggagacctg gtcaagcctggggcatccct gacactcacc tgcacagcct ctggattctccttcagtgcc ggctattgga tatgttgggt ccgccaggctccagggaagg ggctggagtg gatcgcatgc acttatgctggtcgtagtgg tagcacttac tacgcgaact gggtgaatggccgattcacc atccccaaaa cctcgtcgac cacggtgactctgcaaatga ccagtctgtc aggcgcggac acggccagctatttctgtgc gagaggtaat gctggtgttg ctgttggtgccttgtggggc ccaggcaccc tggtcaccgt ctcgagt CDRL1 SEQ ID NO: 95 QASQSISNWLACDRL2 SEQ ID NO: 96 QASKLAS CDRL3 SEQ ID NO: 97 QSYYDSGSNVFFA CDRH1SEQ ID NO: 98 GFSFSAGYWIC CDRH2 SEQ ID NO: 99 CTYAGRSGSTYYANWVNG CDRH3SEQ ID NO: 100 GNAGVAVGAL

The disclosure also extends to a derivative of SEQ ID NO: 97 wherein atleast one of the amino acids in the motif DS is replaced by anotheramino acid, for example the motif is mutated to DA or DT

The disclosure also extends to a derivative of SEQ ID NO: 98 whereincysteine is replaced by another amino acid, for example serine.

The disclosure also extends to a derivative of SEQ ID NO: 99 whereincysteine is replaced by another amino acid, for example serine.

The disclosure also extends to a derivative of SEQ ID NO: 99 wherein atleast one of the amino acids in the motif NG is replaced by anotheramino acid, for example the motif is mutated to NA, NS or NT.

Ab 4129 Rabbit Ab 4129 VL region  SEQ ID NO: 101DIVMTQTPAS VEAAVGGTVT INCQASQSIS SWLSWYQQKPGQPPKLLIYG ASNLASGVPS RFSGSGSGTQ FSLTISDLECADAATYYCQS YYDSGSSVFF NFGGGTKVVV K Rabbit Ab 4129 VL regionSEQ ID NO: 102 gacattgtga tgacccagac tccagcctcc gtggaggcagctgtgggagg cacagtcacc atcaattgcc aagccagtcagagcattagc agttggttat cctggtatca gcagaaaccagggcagcctc ccaagctcct gatctatggt gcatccaatctggcatctgg ggtcccatca cggttcagcg gcagtggatctgggacacag ttcagtctca ccatcagcga cctggagtgtgccgatgctg ccacttacta ctgtcaaagc tattatgatagtggtagtag tgtttttttt aatttcggcg gagggaccaa ggtggtcgtc aaaRabbit Ab 4129 VH region SEQ ID NO: 103QSLEESGGDL VKPGASLTLT CTASGFSFSA GYWICWVRQAPGKGLEWIAC IYAGSSGSTY YASWAKGRFT IPKTSSTTVTLQMTSLTGAD TATYFCARGN AGVAVGALWG PGTLVTVSS Rabbit Ab 4129 VH regionSEQ ID NO: 104 cagtcgttgg aggagtccgg gggagacctg gttaagcctggggcatccct gacactcacc tgcacagcct ctggattctccttcagtgcc ggctattgga tatgttgggt ccgccaggctccagggaagg ggctggagtg gatcgcatgc atttatgctggtagtagtgg tagcacttac tacgcgagct gggcgaaaggccgattcacc atccccaaaa cctcgtcgac cacggtgactctgcaaatga ccagtctgac aggcgcggac acggccacctatttctgtgc gagaggtaat gctggtgttg ctgttggtgccttgtggggc ccaggcaccc tcgtcaccgt ctcgagt CDRL1 SEQ ID NO: 105QASQSISSWLS CDRL2 SEQ ID NO: 106 GASNLAS CDRL3 SEQ ID NO: 107QSYYDSGSSVFFN CDRH1 SEQ ID NO: 108 GFSFSAGYWIC CDRH2 SEQ ID NO: 109CIYAGSSGSTYYASWAKG CDRH3 SEQ ID NO: 110 GNAGVAVGAL

The disclosure also extends to a derivative of SEQ ID NO: 107 wherein atleast one of the amino acids in the motif DS is replaced by anotheramino acid, for example the motif is mutated to DA or DT.

The disclosure also extends to a derivative of SEQ ID NO: 108 whereincysteine is replaced by another amino acid, for example serine.

The disclosure also extends to a derivative of SEQ ID NO: 109 whereincysteine is replaced by another amino acid, for example serine.

Ab 4131 Rabbit Ab 4131 VL region SEQ ID NO: 111DIVMTQTPAS VSEPVGGSVT IKCQASQSFY NLLAWYQQKPGQPPKLLIYD ASDLASGVPS RFKGSGSGTD FTLTISDLECADAAAYYCQS ADGSSYAFGG GTEVVVK Rabbit Ab 4131 VL region SEQ ID NO: 112gacattgtga tgacccagac tccagcctcc gtgtctgaacctgtgggagg ctcagtcacc atcaagtgcc aggccagtcagagcttttac aacctcttag cctggtatca gcagaaaccagggcagcctc ccaaactcct gatctatgat gcatccgatctggcatctgg ggtcccatcg cggttcaaag gcagtggatctgggactgat ttcactctca ccatcagcga cctggagtgtgccgatgctg ccgcttacta ctgtcaaagt gctgatggtagtagttacgc tttcggcgga gggaccgagg tggtcgtcaa a Rabbit Ab 4131 VH regionSEQ ID NO: 113 QEQLEESGGG LVKPEGSLTL TCTASGVSFS SSYWIYWVRQAPGKGLEWIA CIYTGSSGST YYASWAKGRF TVSETSSTTVTLQMTSLTAA DTATYFCARA SAWTYGMDLW GPGTLVTVSS Rabbit Ab 4131 VH regionSEQ ID NO: 114 caggagcaat tggaggagtc cgggggaggc ctggtcaagcctgagggatc cctgacactc acctgcacag cctctggagtctccttcagt agcagctatt ggatatactg ggtccgccaggctccaggga aggggctgga gtggatcgca tgcatttatactggtagtag tggtagcact tactacgcga gctgggcgaaaggccgattc accgtctccg aaacctcgtc gaccacggtgactctgcaaa tgaccagtct gacagccgcg gacacggccacctatttctg tgcgagagca agcgcttgga cctacggcatggacctctgg ggcccgggca ccctcgtcac cgtctcgagt CDRL1 SEQ ID NO: 115QASQSFYNLLA CDRL2 SEQ ID NO: 116 DASDLAS CDRL3 SEQ ID NO: 117 QSADGSSYACDRH1 SEQ ID NO: 118 GVSFSSSYWIY CDRH2 SEQ ID NO: 119 CIYTGSSGSTYYASWAKGCDRH3 SEQ ID NO: 120 ASAWTYGMDL

The disclosure also extends to a derivative of SEQ ID NO: 117 wherein atleast one of the amino acids in the motif DS is replaced by anotheramino acid, for example the motif is mutated to DA or DT.

The disclosure also extends to a derivative of SEQ ID NO: 119 whereincysteine is replaced by another amino acid, for example serine.

Ab 4133 Rabbit Ab 4133 VL region SEQ ID NO: 121AQVLTQTPSP VSAVVGGTVS ISCQASQSVY NNNNLSWYQQKPGQPPKLLI YDASKLASGV PSRFKGSGSG TQFTLTISGVQCDDAATYYC LGGYYSSGWY FAFGGGTKVV VK Rabbit Ab 4133 VL regionSEQ ID NO: 122 gcgcaagtgc tgacccagac tccatctccc gtgtctgcagttgtgggagg cacagtcagc atcagttgcc aggccagtcagagtgtttat aataacaaca acttatcctg gtatcagcagaaaccagggc agcctcccaa gctcttgatc tacgatgcatccaaattggc atctggggtc ccatcccggt tcaaaggcagtggatctggg acacagttca ctctcaccat cagcggcgtgcagtgtgacg atgctgccac ttactactgt ctaggcggttattatagtag tggttggtat tttgctttcg gcggagggac caaggtggtg gtcaaaRabbit Ab 4133 VH region SEQ ID NO: 123QEQLVESGGG LVQPEGSLTL TCTASGFSFS GNYYMCWVRQAPGKGLEWIG CLYTGSSGST YYASWAKGRF TISKTSSTTVTLQMTSLTAA DTATYFCARD LGYEIDGYGG LWGQGTLVTV SS Rabbit Ab 4133 VH regionSEQ ID NO: 124 caggagcagc tggtggagtc cgggggaggc ctggtccagcctgagggatc cctgacacta acctgcacag cttctggattctccttcagt ggcaactact acatgtgctg ggtccgccaggctccaggga aggggctgga gtggatcgga tgcctttatactggtagtag tggtagcaca tattacgcga gctgggcgaaaggccgattc accatctcca aaacctcgtc gaccacggtgactctgcaaa tgaccagtct gacagccgcg gacacggccacctatttctg tgcgagagat ctaggttatg aaattgatggttatgggggc ttgtggggcc agggcaccct cgtcaccgtc tcgagt CDRL1 SEQ ID NO: 125QASQSVYNNNNLS CDRL2 SEQ ID NO: 126 DASKLAS CDRL3 SEQ ID NO: 127LGGYYSSGWYFA CDRH1 SEQ ID NO: 128 GFSFSGNYYMC CDRH2 SEQ ID NO: 129CLYTGSSGSTYYASWAKG CDRH3 SEQ ID NO: 130 DLGYEIDGYGGL

The disclosure also extends to a derivative of SEQ ID NO: 125 whereinthe glycosylation site NLS is removed, for example is mutated to SLS orQLS.

The disclosure also extends to a derivative of SEQ ID NO: 128 whereincysteine is replaced by another amino acid, for example serine.

The disclosure also extends to a derivative of SEQ ID NO: 130 wherein atleast one of the amino acids in the motif DG is replaced by anotheramino acid, for example the motif is mutated to EG, DA or DS.

Serum Albumin Binding Antibodies

CDRH1 dAbH1 SEQ ID NO: 131 Gly Ile Asp Leu Ser Asn Tyr Ala Ile AsnCDRH2 dAbH1 SEQ ID NO: 132Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys GlyCDRH3 dAbH1 SEQ ID NO: 133Thr Val Pro Gly Tyr Ser Thr Ala Pro Tyr Phe Asp Leu CDRL1 dAbL1SEQ ID NO: 134 Gln Ser Ser Pro Ser Val Trp Ser Asn Phe Leu SerCDRL2 dAbL1 SEQ ID NO: 135 Glu Ala Ser Lys Leu Thr Ser CDRL3 dAbL1SEQ ID NO: 136 Gly Gly Gly Tyr Ser Ser Ile Ser Asp Thr ThrHeavy chain variable domain of anti-albumin antibody (no ds)SEQ ID NO: 137 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu ValGln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala ValSer Gly Ile Asp Leu Ser Asn Tyr Ala Ile Asn TrpVal Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp IleGly Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr AlaThr Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg AspAsn Ser Lys Asn Thr Val Tyr Leu Gln Met Asn SerLeu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys AlaArg Thr Val Pro Gly Tyr Ser Thr Ala Pro Tyr PheAsp Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser SerHeavy chain variable domain of anti-albumin antibody (ds) SEQ ID NO: 138Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu ValGln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala ValSer Gly Ile Asp Leu Ser Asn Tyr Ala Ile Asn TrpVal Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp IleGly Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr AlaThr Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg AspAsn Ser Lys Asn Thr Val Tyr Leu Gln Met Asn SerLeu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys AlaArg Thr Val Pro Gly Tyr Ser Thr Ala Pro Tyr PheAsp Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser SerLight chain variable domain of anti-albumin antibody (no ds)SEQ ID NO: 139 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val SerAla Ser Val Gly Asp Arg Val Thr Ile Thr Cys GlnSer Ser Pro Ser Val Trp Ser Asn Phe Leu Ser TrpTyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu LeuIle Tyr Glu Ala Ser Lys Leu Thr Ser Gly Val ProSer Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp PheThr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp PheAla Thr Tyr Tyr Cys Gly Gly Gly Tyr Ser Ser IleSer Asp Thr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg ThrLight chain variable domain of anti-albumin antibody (ds) SEQ ID NO: 140Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val SerAla Ser Val Gly Asp Arg Val Thr Ile Thr Cys GlnSer Ser Pro Ser Val Trp Ser Asn Phe Leu Ser TrpTyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu LeuIle Tyr Glu Ala Ser Lys Leu Thr Ser Gly Val ProSer Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp PheThr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp PheAla Thr Tyr Tyr Cys Gly Gly Gly Tyr Ser Ser IleSer Asp Thr Thr Phe Gly Cys Gly Thr Lys Val Glu Ile Lys Arg ThrHuman CD79a SEQ ID NO: 141 MPGGPGVLQA LPATIFLLFL LSAVYLGPGC QALWMHKVPASLMVSLGEDA HFQCPHNSSN NANVTWWRVL HGNYTWPPEFLGPGEDPNGT LIIQNVNKSH GGIYVCRVQE GNESYQQSCGTYLRVRQPPP RPFLDMGEGT KNRIITAEGI ILLFCAVVPGTLLLFRKRWQ NEKLGLDAGD EYEDENLYEG LNLDDCSMYE DISRGLQGTY QDVGSLNIGD VQLEKPHuman CD79b SEQ ID NO: 142 MARLALSPVP SHWMVALLLL LSAEPVPAAR SEDRYRNPKGSACSRIWQSP RFIARKRGFT VKMHCYMNSA SGNVSWLWKQEMDENPQQLK LEKGRMEESQ NESLATLTIQ GIRFEDNGIYFCQQKCNNTS EVYQGCGTEL RVMGFSTLAQ LKQRNTLKDGIIMIQTLLII LFIIVPIFLL LDKDDSKAGM EEDHTYEGLDIDQTATYEDI VTLRTGEVKW SVGEHPGQE Human CD45 SEQ ID NO: 143MYLWLKLLAF GFAFLDTEVF VTGQSPTPSP TGLTTAKMPSVPLSSDPLPT HTTAFSPAST FERENDFSET TTSLSPDNTSTQVSPDSLDN ASAFNTTGVS SVQTPHLPTH ADSQTPSAGTDTQTFSGSAA NAKLNPTPGS NAISDVPGER STASTFPTDPVSPLTTTLSL AHHSSAALPA RTSNTTITAN TSDAYLNASETTTLSPSGSA VISTTTIATT PSKPTCDEKY ANITVDYLYNKETKLFTAKL NVNENVECGN NTCTNNEVHN LTECKNASVSISHNSCTAPD KTLILDVPPG VEKFQLHDCT QVEKADTTICLKWKNIETFT CDTQNITYRF QCGNMIFDNK EIKLENLEPEHEYKCDSEIL YNNHKFTNAS KIIKTDFGSP GEPQIIFCRSEAAHQGVITW NPPQRSFHNF TLCYIKETEK DCLNLDKNLIKYDLQNLKPY TKYVLSLHAY IIAKVQRNGS AAMCHFTTKSAPPSQVWNMT VSMTSDNSMH VKCRPPRDRN GPHERYHLEVEAGNTLVRNE SHKNCDFRVK DLQYSTDYTF KAYFHNGDYPGEPFILHHST SYNSKALIAF LAFLIIVTSI ALLVVLYKIYDLHKKRSCNL DEQQELVERD DEKQLMNVEP IHADILLETYKRKIADEGRL FLAEFQSIPR VFSKFPIKEA RKPFNQNKNRYVDILPYDYN RVELSEINGD AGSNYINASY IDGFKEPRKYIAAQGPRDET VDDFWRMIWE QKATVIVMVT RCEEGNRNKCAEYWPSMEEG TRAFGDVVVK INQHKRCPDY IIQKLNIVNKKEKATGREVT HIQFTSWPDH GVPEDPHLLL KLRRRVNAFSNFFSGPIVVH CSAGVGRTGT YIGIDAMLEG LEAENKVDVYGYVVKLRRQR CLMVQVEAQY ILIHQALVEY NQFGETEVNLSELHPYLHNM KKRDPPSEPS PLEAEFQRLP SYRSWRTQHIGNQEENKSKN RNSNVIPYDY NRVPLKHELE MSKESEHDSDESSDDDSDSE EPSKYINASF IMSYWKPEVM IAAQGPLKETIGDFWQMIFQ RKVKVIVMLT ELKHGDQEIC AQYWGEGKQTYGDIEVDLKD TDKSSTYTLR VFELRHSKRK DSRTVYQYQYTNWSVEQLPA EPKELISMIQ VVKQKLPQKN SSEGNKHHKSTPLLIHCRDG SQQTGIFCAL LNLLESAETE EVVDIFQVVKALRKARPGMV STFEQYQFLY DVIASTYPAQ NGQVKKNNHQEDKIEFDNEV DKVKQDANCV NPLGAPEKLP EAKEQAEGSE PTSGTEGPEH SVNGPASPAL NQGS

REFERENCES

-   1. Ribosome display efficiently selects and evolves high-affinity    antibodies in vitro from immune libraries. Hanes J, Jermutus L,    Weber-Bornhauser S, Bosshard H R, Plückthun A. (1998) Proc. Natl.    Acad. Sci. U.S.A. 95, 14130-14135-   2. Directed in Vitro Evolution and Crystallographic Analysis of a    Peptide-binding Single Chain Antibody Fragment (scFv) with Low    Picomolar Affinity. Zhand C, Spinelli S, Luginbuhl B, Amstutz P,    Cambillau C, Pluckthun A. (2004) J. Biol. Chem. 279, 18870-18877-   3. Antigen recognition by conformational selection. Berger C,    Weber-Bornhauser S, Eggenberger Y, Hanes J, Pluckthun A,    Bosshard H. R. (1999) F.E.B.S. Letters 450, 149-153

EXAMPLES

The term Fab-Kd-Fab as used in the Examples describes the bispecificprotein complex having the formula A-X:Y-B wherein:

-   -   A-X is a first fusion protein;    -   Y-B is a second fusion protein;    -   X:Y is a heterodimeric-tether;    -   A comprises a Fab fragment specific to an antigen such as CD45        or CD79;    -   B comprises a Fab fragment specific to an antigen such as CD45        or CD79;    -   X is a first binding partner of a binding pair such as a scFv;    -   Y is a second binding partner of the binding pair such as a        peptide; and    -   : is an interaction (such as a binding interaction) between X        and Y.

Example 1—Production of Fab-A (Fab-scFv [A-X]) and Fab-B (Fab-Peptide[B-Y) for Functional Assays Cloning Strategy

Antibody variable region DNA was generated by PCR or gene synthesisflanking restriction enzyme sites DNA sequence. These sites were HindIIIand XhoI for variable heavy chains and HindIII and BsiWI for variablelight chains. This makes the heavy variable region amenable to ligatinginto the two heavy chain vectors (pNAFH with FabB-Y and pNAFH withFabA-Xds [disulphide stabilised]) as they have complementary restrictionsites. This ligates the variable region upstream (or 5′) to the murineconstant regions and peptide Y (GCN4) or scFv X (52SR4) creating a wholereading frame. The light chains were cloned into standard in housemurine constant kappa vectors (pMmCK or pMmCK S171C) which again use thesame complimentary restriction sites. The pMmCK S171C vector is used ifthe variable region is isolated from a rabbit. The cloning events wereconfirmed by sequencing using primers which flank the whole open readingframe.

Cultivating CHOS

Suspension CHOS cells were pre-adapted to CDCHO media (Invitrogen)supplemented with 2 mM (100×) glutamx. Cells were maintained inlogarithmic growth phase agitated at 140 rpm on a shaker incubator(Kuner AG, Birsfelden, Switzerland) and cultured at 37° C. supplementedwith 8% CO₂.

Electroporation Transfection

Prior to transfection, the cell numbers and viability were determinedusing CEDEX cell counter (Innovatis AG. Bielefeld, Germany) and requiredamount of cells (2×10⁸ cells/ml) were transferred into centrifugeconical tubes and were spun at 1400 rpm for 10 minutes. The Pelletedcells were re-suspended in sterile Earls Balanced Salts Solution andspun at 1400 rpm for further 10 minutes. Supernatant was discarded andpellets were re-suspended to desired cell density.

Vector DNA at a final concentration of 400 ug for 2×10⁸ cells/ml mix and800 μl was pipetted into Cuvettes (Biorad) and electroporated usingin-house electroporation system.

Transfected cells were transferred directly into 1×3 L Erlenmeyer Flaskscontained ProCHO 5 media enriched with 2 mM glutmax and antibioticantimitotic (100×) solution (1 in 500) and Cells were cultured in Kuhnershaker incubator set at 37° C., 5% CO₂ and 140 rpm shaking. Feedsupplement 2 g/L ASF (AJINOMOTO) was added at 24 hr post transfectionand temperature dropped to 32° C. for further 13 days culture. At dayfour 3 mM Sodium buryrate (n-BUTRIC ACID Sodium Salt, Sigma B-5887) wasadded to the culture.

On day 14, cultures were transferred to tubes and supernatant separatedfrom the cells after centrifugation for 30 minutes at 4000 rpm. Retainedsupernatants were further filtered through 0.22 um SARTO BRAN PMillipore followed by 0.22 μm Gamma gold filters. Final expressionlevels were determined by Protein G-HPLC.

Large Scale (1.0 L) Purification

The Fab-A and Fab-B were purified by affinity capture using the AKTAXpress systems and HisTrap Excel pre-packed nickel columns (GEHealthcare). The culture supernatants were 0.22 μm sterile filtered andpH adjusted to neutral, if necessary, with weak acid or base beforeloading onto the columns. A secondary wash step, containing 15-25 mMImidazole, was used to displace any weakly bound host cellproteins/non-specific His binders from the nickel resin. Elution wasperformed with 10 mM sodium phosphate, pH7.4+1M NaCl+250 mM Imidazoleand 2 ml fractions collected. One column volume into the elution thesystem was paused for 10 minutes to tighten the elution peak, andconsequently decrease the total elution volume. The cleanest fractionswere pooled and buffer exchanged into PBS (Sigma), pH7.4 and 0.22 μmfiltered. Final pools were assayed by A280 Scan, SE-HPLC (G3000 method),SDS-PAGE (reduced & non-reduced) and for endotoxin using the PTSEndosafe system.

Example 2—CD45 Fab/CD79Fab Bispecific Complex but not a Mixture of CD45and CD79 Fab or Bivalent CD79 Fab Complex Inhibits Akt Signalling

Human PBMC derived from platelet apheresis cones were banked as frozenaliquots. Prior to an assay being performed, cells were thawed, washedin DMEM (Life Technologies) and allowed to acclimatise to a 37° C./5%CO₂ environment. During this period grids of bispecific or bivalentantibodies were created by diluting equimolar (200 nM) quantities ofFab′-A (Fab-scFv) and Fab-B (Fab-peptide) or Fab-A (Fab-peptide) andFab-B (Fab-peptide) with antigen specificity for the cell surfaceproteins CD45 and CD79b in DMEM containing 10% calf serum and 2 mMglutamine. This grid is shown in Table 4.

TABLE 4 Grid of bispecific and bivalent combinations of antibodies withspecificity for CD45 and CD79b. (A-X or Y) (B-Y) Fab B Fab A CD45-YCD79b-Y CD45-X CD45-X:Y-CD45 CD45-X:Y-CD79b CD79b-X CD79b-X:Y-CD45CD79b-X:Y-CD79b CD45-Y CD45-Y:CD79b-Y where X is a scFv (52SR4) and Y isa peptide (GCN4)

FabA-X and FabB-Y or Fab-A-Y and Fab-B-Y were incubated together for 90minutes (in a 37° C./5% CO₂ environment) before mixing with 2.5×10⁵ PBMCin V bottomed 96 well plates. PBMC plus bispecific or bivalentcombinations were then incubated together for a further 90 minutes.After this time B cells were activated by the addition of 200 nM of goatF(ab′)₂ anti-human IgM (Southern Biotechnology) for 8 minutes at 37° C.The signalling reaction was then halted by adding an equal volume ofCytofix buffer (BD Biosciences). Plates were then left at roomtemperature for 15 minutes before centrifugation at 500 g for 5 minutes.Excess supernatant was discarded from the cell pellet which wasresuspended in flow buffer (PBS+1% BSA+0.01% NaN₃) and washed once more.Cells were then resuspended in ice cold Perm Buffer III (BD Biosciences)for 30 minutes before being washed twice in flow buffer. Cells were thenstained with a fluorescently labelled anti-CD20 antibody (BDBiosciences) and a fluorescently labelled anti-phospho Akt antibody thatrecognises a modified serine residue at position 473 on the protein.Plates were then resuspended and incubated for 1 hour at roomtemperature in the dark. After this time plates were washed a furthertwo times and resuspended in 25 μl of flow buffer. Cellular expressionof CD20 and Akt was measured using an Intellicyt HTFC flow cytometer.

Using the data analysis software package FORECYT (Intellicyt) B cellswere identified as distinct from other cell populations and thegeometric mean of Akt levels was calculated for each well. All data wasthen expressed as the percentage inhibition of the maximal response(anti-IgM only) minus the background (cells only). The relative effectof the combinations of CD45 and CD79b is shown in Table 5 (↓=inhibition,↑=stimulation and ↔=no overall effect).

TABLE 5 Table of the relative potency of inhibition of phosphorylatedAkt for bispecific & bivalent combinations of antibodies withspecificity for CD45 & CD79b. (A-X) (B-Y) Fab B Fab A CD45-Y CD79b-YCD45-X Not Tested Not Tested CD79b-X ↓↓ ↔ CD45-Y Not tested ↔where X is a scFv (52SR4) and Y is a peptide (GCN4).

This data is also shown in the form of a bar chart (FIG. 1): the datarepresents mean values and the error bars are 95% confidence intervals.The data shows that the bispecific combination of CD45 with CD79b caninhibit phospho-Akt expression in B cells stimulated with anti-IgM,whereas combining CD79b-Y with CD79b-Y, which is a mixture which cannotform a bispecific, does not.

Example 3—CD45 Fab/CD79Fab Bispecific Complex but not a Mixture of CD45and CD79 Fab or Bivalent CD79 Fab′ Complex Inhibits PLCγ2 Signalling

Human PBMC derived from platelet apheresis cones were banked as frozenaliquots. Prior to an assay being performed cells were thawed, washed inDMEM (Life Technologies) and allowed to acclimatise to a 37° C. /5% CO₂environment. During this period grids of bispecific or bivalentantibodies were created by diluting equimolar (200 nM) quantities ofFab-a (Fab-scFv [A-X]) and Fab′-B (Fab-peptide [B-Y]) or Fab-A(Fab-peptide) and Fab-B (Fab-peptide with antigen specificity for thecell surface proteins CD45 and CD79b in DMEM containing 10% calf serumand 2 mM glutamine. This grid is shown in Table 4.

Fab′A-X and Fab′B-Y or Fab-A-Y and Fab-B-Y were incubated together for90 minutes (in a 37° C./5% CO₂ environment) before mixing with 2.5×10⁵PBMC in V bottomed 96 well plates. PBMC plus bispecific or bivalentcombinations were then incubated together for a further 90 minutes.After this time B cells were activated by the addition of 200 nM of goatF(ab′)2 anti-human IgM (Southern Biotechnology) for 8 minutes at 37° C.The signalling reaction was then halted by adding an equal volume ofCytofix buffer (BD Biosciences). Plates were then left at roomtemperature for 15 minutes before centrifugation at 500 g for 5 minutes.Excess supernatant was discarded from the cell pellet which wasresuspended in flow buffer and washed once more. Cells were thenresuspended in ice cold Perm Buffer III (BD Biosciences) for 30 minutesbefore being washed twice in flow buffer.

Cells were then stained with a fluorescently labelled anti-CD20 antibody(BD Biosciences) and a fluorescently labelled anti-phospho PLCγ2antibody that recognises a modified tyrosine residue at position 759 onthe protein. Plates were then resuspended and incubated for 1 hour atroom temperature in the dark. After this time plates were washed afurther two times and resuspended in 25 μl of flow buffer. Cellularexpression of CD20 and PLCg2 was measured using an Intellicyt HTFC flowcytometer.

Using the data analysis software package FORECYT (Intellicyt) B cellswere identified as distinct from other cell populations and thegeometric mean of PLCγ2 levels was calculated for each well. All datawas then expressed as the percentage inhibition of the maximal response(anti-IgM only) minus the background (cells only). The relative effectof the combination of CD45 and CD79b is shown in Table 6 (↓=inhibition,↑=stimulation and ↔=no overall effect).

TABLE 6 Table of the relative potency of inhibition of phosphorylatedPLCg2 for bispecific and bivalent combinations of antibodies withspecificity for CD45 and CD79b. (A-X or Y) (B-Y) Fab B Fab A CD45-YCD79b-Y CD45-X Not Tested Not Tested CD79b-X ↓↓↓ ↔ CD45-Y Not tested ↔where X is a scFv and Y is a peptide

This data can also be expressed as a bar chart (FIG. 2), the datarepresents mean values and the error bars are 95% confidence intervals.The data shows that the bispecific combination of CD45 with CD79b,inhibit phospho-PLCγ2 expression in B cells stimulated with anti-IgM,whereas combining CD79b-Y with CD79b-Y, which is a mixture which cannotform a bispecific, does not.

Example 4 the Bispecific CD45 and CD79b Complex can Potently Inhibit theExpression of CD86 on B Cells

Human PBMC derived from platelet apheresis cones were banked as frozenaliquots. Prior to an assay being performed cells were thawed, washed inDMEM (Life Technologies) and allowed to acclimatise to a 37 degree C./5%CO₂ environment. During this period bispecific combinations were createdby diluting equimolar (500 nM) quantities of Fab-X (Fab-scFv) and Fab-Y(Fab-peptide) with antigen specificity for the cell surface proteinsCD45 and CD79b in DMEM containing 10% calf serum and 2 mM glutamine.These combinations were then diluted in 8 stepwise 1 in 2.5 dilutions tocreate a dose titration for this combination. Fab-X and Fab-Y wereincubated together for 90 minutes (in a 37 degree C./5% CO₂ environment)before adding 2.5×105 PBMC to V bottomed 96 well plates. PBMC were thenadded to Fab′-X and Fab′-Y combinations and incubated together for afurther 90 minutes. After this time B cells were activated by theaddition of 200 nM of goat F(ab′)2 anti-human IgM (SouthernBiotechnology) for 24 hours at 37 degrees C. To enable detection of cellsurface activation markers plates were placed on ice and washed once inice cold flow buffer (PBS+1% BSA+0.01% NaN3). Cells were then stainedwith a fluorescently labelled anti-CD19 antibody (BD Biosciences) and afluorescently labelled anti-CD86 antibody and incubated on ice for 1hour in the dark. After this time plates were washed a further two timesand resuspended in 25 ul of flow buffer. Cellular expression of CD19 andCD86 was measured using an Intellicyt HTFC flow cytometer. Using thedata analysis software package FORECYT (Intellicyt) B cells wereidentified as distinct from other cell populations and the geometricmean of CD86 levels was calculated for each well. All data was thenexpressed as the percentage inhibition of the maximal response (anti-IgMonly) minus the background (cells only). As can be seen in FIG. 3 atitration of the combination of CD45-X/CD79b-Y was able to inhibitanti-IgM induced CD86 expression on B cells after 24 hours. The IC50, asextrapolated using a 4 parameter logistic curve fit using Graphpad Prism6, was 4.7 nM (the data represents mean values and the error bars arestandard deviations).

Example 5—the Inhibitory Effect of CD45 and CD79b Bispecific Protein canbe Reproduced with Different Antibody V Regions

Immunisation:

DNA encoding antigens CD79a and CD79b and CD45 was obtained by genesynthesis or commercial sources & cloned into an expression vector witha strong constitutive promoter. Plasmid DNA was then transfected intoRab-9 rabbit fibroblast cells (ATCC CRL-1414) using an in-houseelectroporation system. For CD79 immunisations, both CD79a and CD79bwere co-transfected. Twenty four hours later cells were checked forantigen expression by flow cytometry & frozen in aliquots in liquidnitrogen until use. Up to 6 antigens were immunised per rabbit by eitherco-expression on the same cell or making mixtures of singly or multipletransfected cells. Rabbits were immunised with 3 doses of cells.

Antibody Discovery:

B cell cultures were prepared using a method similar to that describedby Zubler et al. (1985). Briefly, spleen or PBMC-derived B cells fromimmunized rabbits were cultured at a density of approximately 2000-5000cells per well in bar-coded 96-well tissue culture plates with 200μl/well RPMI 1640 medium (Gibco BRL) supplemented with 10% FCS (PAAlaboratories ltd), 2% HEPES (Sigma Aldrich), 1% L-Glutamine (Gibco BRL),1% penicillin/streptomycin solution (Gibco BRL), 0.1% β-mercaptoethanol(Gibco BRL), 3% activated splenocyte culture supernatant andgamma-irradiated mutant EL4 murine thymoma cells (5×10⁴/well) for sevendays at 37° C. in an atmosphere of 5% CO₂.

The presence of antigen-specific antibodies in B cell culturesupernatants was determined using a homogeneous fluorescence-basedbinding assay using HEK293 cells co-transfected with CD79a and CD79b orCD45. Screening involved the transfer of 10 ul of supernatant frombarcoded 96-well tissue culture plates into barcoded 384-wellblack-walled assay plates containing HEK293 cells transfected withtarget antigen (approximately 3000 cells/well) using a Matrix Platemateliquid handler. Binding was revealed with a goat anti-rabbit IgGFcγ-specific Cy-5 conjugate (Jackson). Plates were read on an AppliedBiosystems 8200 cellular detection system.

Following primary screening, positive supernatants were consolidated on96-well bar-coded master plates using an Aviso Onyx hit-picking robotand B cells in cell culture plates frozen at −80° C. Master plates werethen screened in a homogeneous fluorescence-based binding assay onHEK293 cells transfected with CD79a and CD79b or CD45 antigens orSUPERAVIDIN beads (Bangs Laboratories) coated with recombinant CD45protein as a source of antigen. This was done in order to determine theantigen specificity for each well.

To allow recovery of antibody variable region genes from a selection ofwells of interest, a deconvolution step was performed to enableidentification of the antigen-specific B cells in a given well thatcontained a heterogeneous population of B cells. This was achieved usingthe Fluorescent foci method (Clargo et al., 2014. Mabs 2014 Jan. 1: 6(1)143-159; EP1570267B1). Briefly, Immunoglobulin-secreting B cells from apositive well were mixed with either HEK293 cells transfected withtarget antigen or streptavidin beads (New England Biolabs) coated withbiotinylated target antigen and a 1:1200 final dilution of a goatanti-rabbit Fcγ fragment-specific FITC conjugate (Jackson). After staticincubation at 37° C. for 1 hour, antigen-specific B cells could beidentified due to the presence of a fluorescent halo surrounding that Bcell. A number of these individual B cell clones, identified using anOlympus microscope, were then picked with an Eppendorf micromanipulatorand deposited into a PCR tube. The fluorescent foci method was also usedto identify antigen-specific B cells from a heterogeneous population ofB cells directly from the bone marrow of immunized rabbits.

Antibody variable region genes were recovered from single cells byreverse transcription (RT)-PCR using heavy and light chain variableregion-specific primers. Two rounds of PCR were performed, with thenested secondary PCR incorporating restriction sites at the 3′ and 5′ends allowing cloning of the variable region into mouse Fab-X and Fab-Y(VH) or mouse kappa (VL) mammalian expression vectors. Heavy and lightchain constructs for the Fab-X and Fab-Y expression vectors wereco-transfected into HEK-293 cells using Fectin 293 (Life Technologies)or Expi293 cells using Expifectamine (Life Technologies) and recombinantantibody expressed in 6-well tissue culture plates in a volume of 5 ml.After 5-7 days expression, supernatants were harvested. Supernatantswere tested in a homogeneous fluorescence-based binding assay on HEK293cells transfected with antigen and SUPERAVIDIN beads (BangsLaboratories) coated with recombinant protein or antigen transfected HEKcells. This was done to confirm the specificity of the clonedantibodies. Production of small scale Fab A-X and Fab B-Y (Small Scale(50 mL) Expi293 Transfection)

The Expi293 cells were routinely sub-cultured in EXPI293 ExpressionMedium to a final concentration of 0.5×10⁶ viable cells/mL and wereincubated in an orbital shaking incubator (Multitron, Infors HT) at 120rpm 8% CO₂ and 37° C.

On the day of transfection cell viability and concentration weremeasured using an automated Cell Counter (Vi-CELL, Beckman Coulter). Toachieve a final cell concentration of 2.5×10⁶ viable cells/mL theappropriate volume of cell suspension was added to a sterile 250 mLErlenmeyer shake flask and brought up to the volume of 42.5 mL by addingfresh, pre-warmed EXPI293 Expression Medium for each 50 mL transfection.

To prepare the lipid-DNA complexes for each transfection a total of 50μg of heavy chain and light chain plasmid DNAs were diluted in OPTI MEMI medium (LifeTechnologies) to a total volume of 2.5 mL and 135 μL ofEXPIFECTAMINE 293 Reagent (LifeTechnologies) was diluted in OPTI MEM Imedium to a total volume of 2.5 mL. All dilutions were mixed gently andincubate for no longer than 5 minutes at room temperature before eachDNA solution was added to the respective diluted EXPIFECTAMINE 293Reagent to obtain a total volume of 5 mL. The DNA-EXPIFECTAMINE 293Reagent mixtures were mixed gently and incubated for 20-30 minutes atroom temperature to allow the DNA-EXPIFECTAMINE 293 Reagent complexes toform.

After the DNA-EXPIFECTAMINE 293 reagent complex incubation wascompleted, the 5 mL of DNA-EXPIFECTAMINE 293 Reagent complex was addedto each shake flask. The shake flasks were incubated in an orbitalshaking incubator (Multitron, Infors HT) at 120 rpm, 8% CO₂ and 37° C.

Approximately 16-18 hours post-transfection, 250 μL of EXPIFECTAMINE 293Transfection Enhancer 1 (LifeTechnologies) and 2.5 mL of EXPIFECTAMINE293 Transfection Enhancer 2 (LifeTechnologies) were added to each shakeflask.

The cell cultures were harvested 7 days post transfection. The cellswere transferred into 50 mL spin tubes (Falcon) and spun down for 30 minat 4000 rpm followed by sterile filtration through a 0.22 um Stericup(Merck Millipore). The clarified and sterile filtered supernatants werestored at 4° C. Final expression levels were determined by ProteinG-HPLC. Small Scale (50 ml) Purification:

Both Fab-X and Fab-Y were purified separately by affinity capture usinga small scale vacuum based purification system. Briefly, the 50 ml ofculture supernatants were 0.22 μm sterile filtered before 500 μL, of NiSepharose beads (GE Healthcare) were added. The supernatant beadsmixture was then tumbled for about an hour before supernatant wasremoved by applying vacuum. Beads were then washed with Wash 1 (50 mMSodium Phosphate 1 M NaCl pH 6.2) and Wash 2 (0.5 M NaCl). Elution wasperformed with 50 mM sodium acetate, pH4.0+1M NaCl. The eluted fractionsbuffer exchanged into PBS (Sigma), pH7.4 and 0.22 μm filtered. Finalpools were assayed by A280 scan, SE-UPLC (BEH200 method), SDS-PAGE(reduced & non-reduced) and for endotoxin using the PTS Endosafe system.

Human PBMC derived from platelet apheresis cones were banked as frozenaliquots. Prior to an assay being performed cells were thawed, washed inRPMI 1640 (Life Technologies) and allowed to acclimatise to a 37° C./5%CO₂ environment. During this period combinations of bispecific, bivalentor mixtures of antibodies were created by diluting equimolar (200 nM)quantities of Fab′-X (Fab-scFv) and Fab′-Y (Fab-peptide) with antigenspecificity for the cell surface proteins CD45 and CD79b in RPMI 1640containing 10% fetal bovine serum, 50 units/mL Penicillin, 50 μg/mLStreptomycin and 2 mM L-glutamine. These combinations of 3 differentCD79b Fab-Ys and 2 different CD45 Fab-Xs are shown in Table 7.

TABLE 7 Grid of bispecific proteins with specificity for CD45 and CD79b.(A-X) (B-Y) Fab B Fab A CD79-Y VR4447 CD79-Y VR4450 CD79b-y VR4246CD45-X CD45-X:Y-CD79b CD45-X:Y-CD79b CD45-X:Y-CD79b VR4131 CD45XCD45-X:Y-CD79b CD45-X:Y-CD79b CD45-X:Y-CD79b VR4248 where X is a scFv(52SR4) and Y is a peptide (GCN4)

FabA-X and FabB-Y were incubated together for 60 minutes (in a 37° C./5%CO₂ environment) before mixing with 2.5×10⁵ PBMC in V bottomed 96 wellplates. PBMC plus FabA-X and/or FabB-Y combinations were then incubatedtogether for a further 90 minutes. After this time B cells wereactivated by the addition of 12.5 μg/mL of goat F(ab′)2 anti-human IgM(Southern Biotechnology) for 10 minutes at 37° C. The signallingreaction was then halted by adding an equal volume of Cytofix buffer (BDBiosciences). Plates were then left at room temperature for 15 minutesbefore centrifugation at 500×g for 5 minutes. Excess supernatant wasdiscarded from the cell pellet which was resuspended in flow buffer(PBS+1% BSA+0.1% NaN₃+2 mM EDTA) and washed once more. Cells were thenresuspended in ice cold Perm Buffer III (BD Biosciences) for 30 minutesbefore being washed twice in flow buffer.

Cells were then stained with a fluorescently labelled anti-CD20 antibody(BD Biosciences), and an anti-phospho PLCγ2 antibody that recognises amodified tyrosine residue at position 759. Plates were then resuspendedand incubated for 1 hour at room temperature in the dark. After thistime plates were washed a further two times and resuspended in 40 μl offlow buffer. Cellular expression of CD20 and PLCγ2 was measured using anIntellicyt HTFC flow cytometer.

Using the data analysis software package FORECYT (Intellicyt) B cellswere identified as distinct from other cell populations and thegeometric mean of PLCγ2 levels were calculated for each well. All datawas then expressed as the percentage inhibition of the maximal response(anti-IgM only) minus the background (cells only).

As can be seen in FIG. 4 the data shows that the combination of CD45with CD79b with different antibody V regions can inhibit phospho-PLCγ2expression in B cells stimulated with anti-IgM.

Example 6: Grid Screening of Large Panels of Heterodimerically TetheredProtein Complexes to Identify Novel Bispecific Antibody Targets

Introduction:

Following the successful validation of the bispecific format andscreening method in the earlier examples the screening was expanded to alarger number of antigen pairs. A panel of antibody variable (V) regionpairs to 23 different antigens expressed on B cells was generated. Usingthe Fab-Kd-Fab [i.e. A-X:Y-B wherein A and B are Fab fragments] format agrid of heterodimerically tethered protein complexes was formedrepresenting multiple V region combinations of each of 315 differentantigen pair combinations. These combinations were screened for theirability to modulate BCR (B cell receptor) signalling in a highthrough-put flow cytometry assay to select novel target pairs forintervention with a bispecific antibody.

Immunisation:

DNA encoding selected antigens was obtained by gene synthesis orcommercial sources & cloned into an expression vector with a strongconstitutive promoter. Plasmid DNA was then transfected into Rab-9rabbit fibroblast cells (ATCC CRL-1414) using an in-houseelectroporation system. Twenty four hours later cells were checked forantigen expression by flow cytometry & frozen in aliquots in liquidnitrogen until use. Up to 6 antigens were immunised per rabbit by eitherco-expression on the same cell or making mixtures of singly or multipletransfected cells. Rabbits were immunised with 3 doses of cells.

Antibody Discovery:

B cell cultures were prepared using a method similar to that describedby Zubler et al. (1985). Briefly, spleen or PBMC-derived B cells fromimmunized rabbits were cultured at a density of approximately 2000-5000cells per well in bar-coded 96-well tissue culture plates with 200μl/well RPMI 1640 medium (Gibco BRL) supplemented with 10% FCS (PAAlaboratories ltd), 2% HEPES (Sigma Aldrich), 1% L-Glutamine (Gibco BRL),1% penicillin/streptomycin solution (Gibco BRL), 0.1% β-mercaptoethanol(Gibco BRL), 3% activated splenocyte culture supernatant andgamma-irradiated mutant EL4 murine thymoma cells (5×10⁴/well) for sevendays at 37° C. in an atmosphere of 5% CO₂.

The presence of antigen-specific antibodies in B cell culturesupernatants was determined using a homogeneous fluorescence-basedbinding assay using HEK293 cells co-transfected with the antigens thatthe rabbits were immunized with. Screening involved the transfer of 10ul of supernatant from barcoded 96-well tissue culture plates intobarcoded 384-well black-walled assay plates containing HEK293 cellstransfected with target antigen (approximately 3000 cells/well) using aMatrix Platemate liquid handler. Binding was revealed with a goatanti-rabbit IgG Fcγ-specific Cy-5 conjugate (Jackson). Plates were readon an Applied Biosystems 8200 cellular detection system.

Following primary screening, positive supernatants were consolidated on96-well bar-coded master plates using an Aviso Onyx hit-picking robotand B cells in cell culture plates frozen at −80° C. Master plates werethen screened in a homogeneous fluorescence-based binding assay onHEK293 cells transfected with antigens separately and SUPERAVIDIN beads(Bangs Laboratories) coated with recombinant protein as a source ofantigen. This was done in order to determine the antigen specificity foreach well.

To allow recovery of antibody variable region genes from a selection ofwells of interest, a deconvolution step was performed to enableidentification of the antigen-specific B cells in a given well thatcontained a heterogeneous population of B cells. This was achieved usingthe Fluorescent foci method (Clargo et al., 2014.Mabs 2014 Jan. 1: 6(1)143-159; EP1570267B1). Briefly, Immunoglobulin-secreting B cells from apositive well were mixed with either HEK293 cells transfected withtarget antigen or streptavidin beads (New England Biolabs) coated withbiotinylated target antigen and a 1:1200 final dilution of a goatanti-rabbit Fcγ fragment-specific FITC conjugate (Jackson). After staticincubation at 37° C. for 1 hour, antigen-specific B cells could beidentified due to the presence of a fluorescent halo surrounding that Bcell. A number of these individual B cell clones, identified using anOlympus microscope, were then picked with an Eppendorf micromanipulatorand deposited into a PCR tube. The fluorescent foci method was also usedto identify antigen-specific B cells from a heterogeneous population ofB cells directly from the bone marrow of immunized rabbits.

Antibody variable region genes were recovered from single cells byreverse transcription (RT)-PCR using heavy and light chain variableregion-specific primers. Two rounds of PCR were performed, with thenested secondary PCR incorporating restriction sites at the 3′ and 5′ends allowing cloning of the variable region into mouse Fab-X and Fab-Y(VH) or mouse kappa (VL) mammalian expression vectors. Heavy and lightchain constructs for the Fab-X and Fab-Y expression vectors wereco-transfected into HEK-293 cells using Fectin 293 (Life Technologies)or Expi293 cells using Expifectamine (Life Technologies) and recombinantantibody expressed in 6-well tissue culture plates in a volume of 5 ml.After 5-7 days expression, supernatants were harvested. Supernatantswere tested in a homogeneous fluorescence-based binding assay on HEK293cells transfected with antigen and SUPERAVIDIN beads (BangsLaboratories) coated with recombinant protein or antigen transfected HEKcells. This was done to confirm the specificity of the clonedantibodies.

Production of small scale Fab A-X and Fab B-Y (Small Scale (50 mL)Expi293 Transfection) The Expi293 cells were routinely sub-cultured inEXPI293 Expression Medium to a final concentration of 0.5×10⁶ viablecells/mL and were incubated in an orbital shaking incubator (Multitron,Infors HT) at 120 rpm 8% CO₂ and 37° C.

On the day of transfection cell viability and concentration weremeasured using an automated Cell Counter (Vi-CELL, Beckman Coulter). Toachieve a final cell concentration of 2.5×10⁶ viable cells/mL theappropriate volume of cell suspension was added to a sterile 250 mLErlenmeyer shake flask and brought up to the volume of 42.5 mL by addingfresh, pre-warmed EXPI293 Expression Medium for each 50 mL transfection.

To prepare the lipid-DNA complexes for each transfection a total of 50μg of heavy chain and light chain plasmid DNAs were diluted in OPTI-MEMI medium (LifeTechnologies) to a total volume of 2.5 mL and 135 μL ofEXPIFECTAMINE 293 Reagent (LifeTechnologies) was diluted in OPTI-MEM Imedium to a total volume of 2.5 mL. All dilutions were mixed gently andincubate for no longer than 5 minutes at room temperature before eachDNA solution was added to the respective diluted EXPIFECTAMINE 293Reagent to obtain a total volume of 5 mL. The DNA-EXPIFECTAMINE 293Reagent mixtures were mixed gently and incubated for 20-30 minutes atroom temperature to allow the DNA-EXPIFECTAMINE 293 Reagent complexes toform.

After the DNA-EXPIFECTAMINE 293 reagent complex incubation wascompleted, the 5 mL of DNA-EXPIFECTAMINE 293 Reagent complex was addedto each shake flask. The shake flasks were incubated in an orbitalshaking incubator (Multitron, Infors HT) at 120 rpm, 8% CO₂ and 37° C.

Approximately 16-18 hours post-transfection, 250 μL of EXPIFECTAMINE 293Transfection Enhancer 1 (LifeTechnologies) and 2.5 mL of EXPIFECTAMINE293 Transfection Enhancer 2 (LifeTechnologies) were added to each shakeflask.

The cell cultures were harvested 7 days post transfection. The cellswere transferred into 50 mL spin tubes (Falcon) and spun down for 30 minat 4000 rpm followed by sterile filtration through a 0.22 um Stericup(Merck Millipore). The clarified and sterile filtered supernatants werestored at 4° C. Final expression levels were determined by ProteinG-HPLC. Small Scale (50 ml) Purification:

Both Fab-X and Fab-Y were purified separately by affinity capture usinga small scale vacuum based purification system. Briefly, the 50 ml ofculture supernatants were 0.22 μm sterile filtered before 500 μL of NiSepharose beads (GE Healthcare) were added. The supernatant beadsmixture was then tumbled for about an hour before supernatant wasremoved by applying vacuum. Beads were then washed with Wash 1 (50 mMSodium Phosphate 1 M NaCl pH 6.2) and Wash 2 (0.5 M NaCl). Elution wasperformed with 50 mM sodium acetate, pH4.0+1M NaCl. The eluted fractionsbuffer exchanged into PBS (Sigma), pH7.4 and 0.22 μm filtered. Finalpools were assayed by A280 scan, SE-UPLC (BEH200 method), SDS-PAGE(reduced & non-reduced) and for endotoxin using the PTS Endosafe system.

Screening Assays

Donor PBMCs were rapidly thawed using a water bath set to 37° C., andcarefully transferred to a 50 ml Falcon tube. They were then diluteddropwise to 5 ml in assay media to minimise the osmotic shock. The cellswere then diluted to 20 ml carefully before adding the final mediadiluent to make the volume 50 ml. The cells were then spun at 500 g for5 minutes before removing the supernatant and resuspending the cells in1 ml media. The cells were then counted and diluted to 1.66×10⁶ cells/mlbefore dispensing 30 μl per well into a V-bottom TC plate giving a finalassay concentration of 5.0×10⁴ cells/well. The cell plate was thenstored covered in a 37° C., 5% CO₂ incubator until they were required,giving them a minimum of 1 hour to rest.

Fab-X and Fab-Y reagents were mixed in an equimolar ratio at 5× thefinal assay concentration in assay media and incubated for 90 min at 37°C., 5% CO₂. Samples were prepared in a 96-well U-bottom polypropyleneplate and covered during the incubation.

10 μl of 5× Fab-KD-Fab mixture was added to the appropriate test wellscontaining cells and mixed by shaking at 1000 rpm for 30 sec prior tobeing incubated for 90 min at 37° C., 5% CO₂. The cells were thenstimulated with 10 μl of anti-human IgM. The final assay concentrationof stimulus varied depending on the assay panel readouts, the threeantibody cocktails A, B and C (detailed below) were stimulated at afinal assay concentration of either 50 μg/ml (cocktail A & C) or 25μg/ml (cocktail B). The assay plates were then gently mixed at 1000 rpmfor 30 sec prior to incubation at 37° C., 5% CO₂ for 5 min (antibodycocktail A & C) or 2 min (antibody cocktail B). The assay was stopped byadding 150 μl ice-cold BD CytoFix to all wells and incubated for 15 minat RT. The fixed cells were then spun at 500 g for 5 min to pellet thecells and allow removal of the supernatant using a BioTek ELx405 platewasher. The pellet was re-suspended by vortexing the plate at 2400 rpmfor 30 sec. The cells were then permeabilised at 4° C. by adding 100 μlice-cold BD Cell Permeabilisation Buffer III for 30 min. The cells werethen washed in 100 μl FACS buffer and spun at 500 g for 5 min.Supernatant was again removed by the ELx405 before using it to rapidlydispense 200 μl FACS Buffer to wash away any residual permeabilisationbuffer. Cells were again spun at 500 g and the supernatant removed byinversion. During the preceding spin step the antibody cocktail wasprepared in FACS Buffer and kept shielded from the light. The cells werethen re-suspended by vortexing (2400 RPM, 30 sec) before 20 μl ofantibody cocktail was added to all wells and the plate shaken for 30 secat 1000 rpm. The cells were then incubated for 60 min at RT in the dark.

The cells were then washed twice in 200 μl FACS buffer with a 500 g spinand supernatant removed after each step. Finally the cells werere-suspended by vortexing for 30 sec at 2400 rpm before adding a final20 μl FACS buffer. The plate(s) were then read on the IntellicytHTFC/iQue instrument.

FACS Buffer=PBS+1% BSA+0.05% NaN₃+2 mM EDTA

Antibody Cocktail A=1:2 CD20 PerCp-Cy5.5 (BD Biosciences)+1:5 PLCγ2AF88+1:10 Akt AF647+1:50 ERK1/2 PE (diluted in FACS buffer).

Antibody Cocktail B=1:2 CD20 PerCp-Cy5.5 (BD Biosciences)+1:5 Syk PE+1:5BLNK AF647 (diluted in FACS buffer)

Antibody Cocktail C=1:5 CD20 PerCp-Cy5.5 (Biolegend)+1:5 PLCγ2AF488+1:10 Akt AF647+1:5 Syk PE (diluted in FACS buffer)

Reagent Supplier Catalogue number Anti-human IgM Southern Biotech2022-14 CytoFix BD Biosciences 554655 Perm Buffer III BD Biosciences558050 Anti Akt (pS473) AF647 BD Biosciences 561670 Anti SYK (pY348) PEBD Biosciences 558529 Anti PLCγ2 (pY759) AF488 BD Biosciences 558507Anti-BLNK(pY84) AF647 BD Biosciences 558443 Anti ERK1/2 (pT202/pY204) PEBD Biosciences 561991 Anti-human CD20 PerCp-Cy5.5 BD Biosciences 558021Anti-human CD20 AF488 BD Biosciences 558056 Anti-human CD20 PerCp-Cy5.5Biolegend 340508 Phosphate Buffer Saline (PBS) Fisher Scientific10562765 RPMI 1640 Life Technologies 31870 Foetal Calf Serum (FCS) LifeTechnologies 16140 Glutamax Life Technologies 35050Penicillin/Streptomycin (P/S) Life Technologies 15070 EDTA Sigma 03690Sodium Azide (NaN3) Sigma S2002 Bovine Serum Albumin (BSA) Sigma A1470

Fab-X+Fab-Y combinations were screened with either antibody cocktail Aand B or C alone. All screens were conducted on cone cells from 2different blood donors. Data was captured and evaluated usingcommercially available software tools. A total of 2500 Fab-X+Fab-Ycombinations were screened to 315 different antigen combinations.

Results

The percentage inhibition of the induction of phosphorylation of BCRsignalling cascade proteins by each Fab-Kd-Fab [i.e. A-X:Y-B where A andB are Fab fragments] combination was calculated, in this example lookingfor new combinations of antigens that inhibit B cell function, thecriteria for a positive combination was set as at least 30% inhibitionof at least two phospho-readouts by at least one combination of Vregions. According to this threshold 11 new antigen pair combinationsout of 315 examined met the required criteria. This represents a 3.5%hit rate demonstrating the importance of screening large numbers ofcombinations to find those of desired activity and how rare the activityof the combination of CD79b and CD45 is.

FIGS. 6-8 show the data for the antigen grid cross specificities. Valuesare percentage inhibition (negative value for activation) ofphosphorlylation of Syk, PLCγ2 & AKT respectively and represent the meanof multiple V-region combinations evaluated. 315 different antigencombinations were tested and as can be seen the effect on BCR signallingby different combinations of antibody varied significantly from stronginhibition e.g. antigen 2 (CD79b) on Fab-X combined with antigen 4(CD45) on Fab-Y (70.4% inhibition of phospho Syk FIG. 6) to activatione.g antigen 6 on X and antigen 11 on Y (minus 118.10% phospho Syk FIG.6). Each data point representing the mean % values represented in FIGS.6-8 is shown for antigen combination 2 (CD79b) on Fab-X and antigen 4(CD45) on Fab-Y in FIG. 9. In this case, 10 different combinations ofdifferent antibody V regions were evaluated. The same antigencombination but in alternative orientation, i.e. antigen 2 (CD79b) onFab-Y and antigen 4 (CD45) on Fab-X is shown in FIG. 10. In this case, 6different combinations of different antibody V regions were evaluated.Again, all V regions show inhibition but optimal V region combinationscan be identified and selected using the method.

Example 7—Screening of Transiently Expressed V-Regions to Antigen CD45as Fab-X with Purified Anti-CD79b Fab-Y in Heterodimerically TetheredProtein Complexes to Select Optimal Anti-CD45 Antibody V-Regions

Introduction:

New V-regions to CD45 that inhibit B cell signalling as a bispecificantibody in combination with CD79b specific V regions were identifiedusing grid screening of heterodimerically tethered protein complexes.The CD45 V regions were expressed transiently as Fab-X and combined withpurified anti-CD79b Fab-Y. The inhibition of activation of B cellsignalling was measured to select the most potent anti-CD45 andanti-CD79b V regions. The preparation of antigen expressing cells andimmunisation of rabbits was carried out in the same way as described inExample 6.

Antibody Discovery:

B cell cultures were prepared in the same way as described in Example 6.The screening of antigen-specific antibodies in B cell culturesupernatants and the deconvolution step for identification of antigenspecific B cells was determined in the same way as Example 6.

Antibody variable region genes were recovered from single cells byreverse transcription (RT)-PCR using heavy and light chain variableregion-specific primers. Two rounds of PCR were performed, with thenested 2° PCR incorporating restriction sites at the 3′ and 5′ endsallowing cloning of the variable region into mouse Fab-X and mouse kappa(VL) mammalian expression vector. These vectors were then co-transfectedin HEK-293 cells using 293Fectin (Life Technologies) or in Expi293 cellsusing Expifectamine (Life Technologies) and left to express for 6 days.Supernatants were tested in a homogeneous fluorescence-based bindingassay on HEK293 cells transfected with antigen and SUPERAVIDIN beads(Bangs Laboratories) coated with recombinant protein or antigentransfected HEK cells. This was done to confirm the specificity of thecloned antibodies.

In addition to the Fab-X transient supernatants, negative control Mocksupernatants were prepared in the same way using an irrelevant controlDNA.

The expression levels of Fab-X were determined by Protein G-HPLC.

Production of Purified Fab-X and Fab-Y:

Purified Fab-X and Fab-Y was prepared using the same method described inExample 6.

PhosFlow Assay:

CD79b-specific Fab-Y and CD45-specific Fab-X, either purified or intransient supernatant, were incubated together for 60 minutes (in a 37°C. & 5% CO₂ environment) at equimolar concentration of 200 nM and 90 nM.A mock supernatant was also included neat. In V-bottomed 96 well plates,5.0×10⁴ PBMC were added to wells, to which were added titrated Fab-X andFab-Y combinations or mock supernatant. The combinations and cells werethen incubated together for a further 90 minutes. After this time Bcells were activated by the addition of 25 μg/mL of goat F(ab′)₂anti-human IgM (Southern Biotechnology) for 15 minutes at 37° C. plus 5%CO₂. The signalling reaction was then halted by adding an equal volumeof Cytofix buffer (BD Biosciences). Plates were then left at roomtemperature for 15 minutes before centrifugation at 500×g for 5 minutes.Excess supernatant was discarded from the cell pellet which wasresuspended in FACS buffer (PBS+1% BSA+0.01% NaN₃+2 mM EDTA) and washedonce more. Cells were then resuspended in ice cold Perm Buffer III (BDBiosciences) for 30 minutes before being washed twice in flow buffer.Cells were then stained as described in Example 6, except that insteadof 3 different antibody cocktails, only one cocktail was used with thesame assay concentrations and incubation conditions as described forantibody cocktail A in Example 6.

Antibody Cocktail=1:3 CD20 PerCp-Cy5.5+1:5 PLCγ2 AF88+1:10 Akt AF647+1:5p38 MAPK PE (diluted in FACS buffer).

Results

As can be seen in FIGS. 11 to 16, the data shows that the combination ofdifferent transiently expressed antigen CD45 V regions in Fab-X with 2different purified antigen CD79b V regions (VR447 and VR4450) in Fab-Ycan inhibit B cell activation (as measured by inhibition of PLCγ2, p38and Akt) to different levels and screening in a bispecific formattherefore facilitates selection of optimal V region combinations.Combinations with transient Fab-X are compared to a referencecombination with a purified CD45 Fab-X (VR4122).

Example 8—Effect of Co-Targeting the Antigen CD79b Plus Antigen CD45 onMemory B Cell Function Using Molecularly Linked Bispecific Bybes with orwithout Further Addition of an Anti-Albumin

Introduction:

To check that targeting CD79b/CD45 has a functional effect on B cells inlong term culture, IgG production from B cells in a mixed PBMC culturewas measured. The measurement of specific antibodies to the recallantigen tetanus toxoid provides a read out of memory B cell function.

Antigen CD79b specificity (VR4447) and antigen CD45 specificity (VR4248and VR4133) were generated in a BYbe format with or without addition ofan anti-albumin fragment (VR0645). The anti-albumin antibody fragmentwas fused to the light chain of the antigen CD45 Fab of the BYbe formatas described in Example 8.

Description of constructs used in this experiment.

Heavy Chain Light Chain Construct Name Fab Specificity scFv sFvVR4447/VR4248 BYbe Antigen CD79b Antigen CD45 None VR4447/VR4248/VR645Antigen CD79b Antigen CD45 Albumin BYbe/Albumin VR4447/VR4133 BYbeAntigen CD79b Antigen CD45 None VR4447/VR4133/VR645 Antigen CD79bAntigen CD45 Albumin BYbe/Albumin

Methods

Purification of BYbes with/without Anti-Albumin Additional Specificity

The BYbe (Fab-dsscFv [scFv off C-terminus of Fab heavy chain]) and BYbewith anti-albimin (Fab-2×dsscFv [scFvs off C-terminus of Fab heavy chainand light chain]) formats were purified as follows. Clarified cellculture supernatants from standard expiHEK or CHO expression were 0.22μm sterile filtered. The filtered supernatants were loaded at 2 ml/minonto 50 ml GammabindPlus Sepharose XK26 columns (GE Healthcare)equilibrated in PBS pH7.4 (Sigma Aldrich Chemicals). After loading thecolumns were washed with PBS pH7.4 and then eluted with 0.1MGlycine/HCl. pH 2.7. The elution was followed by absorbance at 280 nm,the elution peak collected, and then neutralised with 1/25^(th) volumeof 2 M Tris/HCl pH8.5. The neutralised samples were concentrated usingAmicon Ultra-15 concentrators with either a 10 kDa or 30 kDa molecularweight cut off membrane and centrifugation at 4000×g in a swing outrotor. Concentrated samples were applied to either a XK16/60 or XK26/60Superdex 200 column (GE Healthcare) equilibrated in PBS, pH7.4. Thecolumns were developed with an isocratic gradient of PBS, pH7.4 ateither 1 ml/min or 2.6 ml/min respectively. Fractions were collected andanalysed by size exclusion chromatography on a TSK gel G3000SWXL; 5 μm,7.8×300 mm column developed with an isocratic gradient of 0.2 Mphosphate, pH 7.0 at 1 ml/min, with detection by absorbance at 280 nm.Selected monomer fractions were pooled and concentrated to >1 mg/mlusing an Amicon Ultra-15 concentrator with a 10 kDa or 30 kDa molecularweight cut off membrane and centrifugation at 4000×g in a swing outrotor. Final samples were assayed; for concentration by A280 ScanningUV-visible spectrophotometer (Cary 50Bio); for % monomer by sizeexclusion chromatography on a TSK gel G3000SWXL; 5 μm, 7.8×300 mm columndeveloped with an isocratic gradient of 0.2 M phosphate, pH7.0 at 1ml/min, with detection by absorbance at 280 nm; by reducing andnon-reducing SDS-PAGE run on 4-20% Tris-Glycine 1.5 mm gels (Novex) at50 mA (per gel) for 53 minutes; and for endotoxin by Charles River'sENDOSAFE Portable Test System with Limulus Amebocyte Lysate (LAL) testcartridges.

Activation of B Cells and Measurement of Tetanus Toxoid Specific IgG

Human PBMCs were stimulated with 500 ng/ml CD40L, 1 ug/ml CpG and 50ng/ml IL-21 in 1640 media plus 10% foetal bovine serum and 2 mM Glutamax(R10 medium) for 6 days. Constructs of purified protein were added at afinal concentration of 100 nM at day 0 and remained in the culturemedium for the duration of the assay. After 6 days the supernatants wereharvested and the amount of tetanus toxoid specific IgG was detected byELISA. Briefly, Maxisorp half-well ELISA plates (Nunc) were coated with10 ug/ml tetanus toxoid in PBS overnight at 4° C. The plates were thenblocked in 5% Milk—in PBS containing 0.05% Tween 20 for 2 hours. Thesupernatants were diluted and then added for 2 hours at roomtemperature. The plates were washed with PBS-0.05% Tween20 and tetanusbound antibody was detected using a peroxidase-goat anti-human IgG(H+L)diluted to 1 ug/ml in 5% milk-PBS 0.05% Tween 20. Plates were developedusing TMB substrate solution (KPL) and absorbance was measured at 450 nMusing a Synergy 2 micro-plate reader (Biotek). Data was exported toExcel and percentage inhibition was calculated relative to cellscultured without test antibodies. The data was then imported intoGraphpad PRISM and plotted as bar charts.

FIG. 17 shows the inhibition of tetanus toxoid IgG production from PBMCscultured with VR4447/VR4248 BYbe, VR4447/VR4133 BYbe,VR4447/VR4248/VR645 BYbe/Albumin and VR4447/VR4133/VR645 BYbe/Albumin.Data shown is from a single donor.

1-33. (canceled)
 34. An antibody comprising a binding domain specific toantigen CD45 and a binding domain specific to antigen CD79b, wherein thebinding domain specific to the antigen CD45 comprises: 3 heavy chainCDRs comprising SEQ ID NO: 128 for CDRH1, SEQ ID NO: 129 for CDRH2 andSEQ ID NO: 130 for CDRH3 and 3 light chain CDRs comprising SEQ ID NO:125 for CDRL1, SEQ ID NO: 126 for CDRL2 and SEQ ID NO: 127 for CDRL3;wherein the binding domain specific to the antigen CD79b comprises: 3heavy chain CDRs comprising SEQ ID NO: 88 for CDRH1, SEQ ID NO: 89 forCDRH2 and SEQ ID NO: 90 for CDRH3 and 3 light chain CDRs comprising SEQID NO: 85 for CDRL1, SEQ ID NO: 86 for CDRL2 and SEQ ID NO: 87, or aderivative thereof, for CDRL3 wherein, in at least one CDR (a) at leastone cysteine residue has been substituted with another amino acid, (b)at least one aspartic acid isomerisation site has been removed bysubstituting at least one amino acid, (c) at least one asparaginedeamidation site has been removed by substituting at least one aminoacid, (d) at least one glycosylation site has been removed bysubstituting at least one amino acid, or (e) a combination thereof.